WO2024137953A2 - Polypeptides de petite ribonucléoprotéine nucléaire 13, polynucléotides et utilisations associées - Google Patents

Polypeptides de petite ribonucléoprotéine nucléaire 13, polynucléotides et utilisations associées Download PDF

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Publication number
WO2024137953A2
WO2024137953A2 PCT/US2023/085370 US2023085370W WO2024137953A2 WO 2024137953 A2 WO2024137953 A2 WO 2024137953A2 US 2023085370 W US2023085370 W US 2023085370W WO 2024137953 A2 WO2024137953 A2 WO 2024137953A2
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seq
amino acid
position corresponding
polypeptide
mrna
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PCT/US2023/085370
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WO2024137953A3 (fr
Inventor
Elizaveta A. ANDRIANOVA
Athanasios DOUSIS
Ruchi Jain
Kanchana RAVICHANDRAN
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Modernatx, Inc.
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Publication of WO2024137953A2 publication Critical patent/WO2024137953A2/fr
Publication of WO2024137953A3 publication Critical patent/WO2024137953A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors

Definitions

  • Snu13 Small nuclear ribonucleoprotein 13 is a nuclear protein that is a component of the [U4/U6.U5] tri-snRNP (small nuclear ribonucleoprotein). Snu13 binds to the 5’ stem-loop of U4 snRNA.
  • the present disclosure provides Snu13 polypeptides and polynucleotides (e.g., mRNA) encoding said Snu13 polypeptides.
  • the present disclosure also provides messenger RNAs (mRNAs) for regulating polypeptide expression.
  • mRNAs messenger RNAs
  • the Snu13 polypeptides and polynucleotides of the disclosure may be used to, e.g., regulate expression of a polypeptide (e.g., in a subject) from a polynucleotide that contains a Snu13-binding site and encodes the polypeptide.
  • the regulation of expression of a polypeptide may be achieved through expression of exogenous Snu13 polypeptide (e.g., a Snu13 polypeptide disclosed herein).
  • Snu13 polynucleotides (e.g., mRNAs) of the invention are particularly well-suited for regulating expression of a polypeptide in a subject, as the technology provides for the intracellular delivery of mRNA encoding a Snu13 polypeptide followed by de novo synthesis of functional Snu13 polypeptide within target cells.
  • the instant invention optionally includes the incorporation of modified nucleotides within mRNAs to (1) minimize unwanted immune activation (e.g., the innate immune response associated with the in vivo introduction of foreign nucleic acids) and (2) optimize the translation efficiency of mRNA to protein.
  • modified nucleotides within mRNAs to (1) minimize unwanted immune activation (e.g., the innate immune response associated with the in vivo introduction of foreign nucleic acids) and (2) optimize the translation efficiency of mRNA to protein.
  • Exemplary aspects of the disclosure feature a combination of nucleotide modification to reduce the innate immune response and sequence optimization, in particular, within the open reading frame (ORF) and untranslated regions (UTRs) of mRNAs encoding a Snu13 polypeptide to enhance protein expression.
  • ORF open reading frame
  • UTRs untranslated regions
  • the Snu13 polypeptide preferentially binds to and represses translation from an mRNA containing a Snu13-binding site and N1- methylpseudouracils (G5).
  • the mRNA technology of the instant disclosure also features delivery of mRNA encoding a Snu13 polypeptide via a lipid nanoparticle (LNP) delivery system.
  • the mRNA technology of the instant disclosure also features delivery of mRNA containing a Snu13-binding site and an open reading frame encoding a polypeptide.
  • compositions and delivery formulations comprising a polynucleotide, e.g., a ribonucleic acid (RNA), e.g., an mRNA, encoding a Snu13 polypeptide and methods for regulating expression of a polypeptide in a human subject in need thereof by administering the same.
  • RNA ribonucleic acid
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a lipid nanoparticle encapsulated mRNA that comprises an ORF encoding a Snu13 polypeptide, wherein the composition is suitable for administration to a human subject in need of regulation of expression of a polypeptide.
  • the disclosure provides a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence comprises: (i) an amino acid other than leucine (L) at the position corresponding to position 35 of SEQ ID NO:1; (ii) an amino acid other than lysine (K) at the position corresponding to position 37 of SEQ ID NO:1; (iii) an amino acid other than alanine (A) at the position corresponding to position 39 of SEQ ID NO:1; (iv) an amino acid other than asparagine (N) at the position corresponding to position 40 of SEQ ID NO:1; (v) an amino acid other than alanine (A) at the position corresponding to position 42 of SEQ ID NO:1; (vi) an amino acid other than aspartic acid (D) at the position corresponding to position 59 of SEQ ID NO:1; (vii) an amino acid other than alanine (A) at
  • the amino acid sequence comprises: (a) (i) an amino acid other than alanine (A) at the position corresponding to position 39 of SEQ ID NO:1, (ii) an amino acid other than asparagine (N) at the position corresponding to position 40 of SEQ ID NO:1, (iii) an amino acid other than glutamic acid (E) at the position corresponding to position 61 of SEQ ID NO:1, (iv) an amino acid other than isoleucine (I) at the position corresponding to position 65 of SEQ ID NO:1, (v) an amino acid other than lysine (K) at the position corresponding to position 86 of SEQ ID NO:1, and (vi) an amino acid other than glutamine (Q) at the position corresponding to position 114 of SEQ ID NO:1; (b) (i) an amino acid other than lysine (K) at the position corresponding to position 37 of SEQ ID NO:1; (ii) an amino acid other than alanine (A)
  • the amino acid sequence comprises: (a) one or more substitutions selected from the group consisting of A39N, N40Y, E61S, I65K, K86S, and Q114H (numbered according to SEQ ID NO:1); (b) one or more substitutions selected from the group consisting of K37R, A39V, N40H, E61H, P62H, I65K, I66L, Attorney Docket No.45817-0124WO1 / MTX959.20 and K86S (numbered according to SEQ ID NO:1); (c) one or more substitutions selected from the group consisting of K37T, A39R, N40H, I65K, I66V, and K86S (numbered according to SEQ ID NO:1); (d) one or more substitutions selected from the group consisting of A39V, N40K, A42V, A60V, E61S, I65V, V95I, S96D, R97M, V99L, A101V, K113S
  • the amino acid sequence comprises: (a) the substitutions A39N, N40Y, E61S, I65K, K86S, and Q114H (numbered according to SEQ ID NO:1); (b) the substitutions K37R, A39V, N40H, E61H, P62H, I65K, I66L, and K86S (numbered according to SEQ ID NO:1); (c) the substitutions K37T, A39R, N40H, I65K, I66V, and K86S (numbered according to SEQ ID NO:1); (d) the substitutions A39V, N40K, A42V, A60V, E61S, I65V, V95I, S96D, R97M, V99L, A101V, K113S, and Q114N (numbered according to SEQ ID NO:1); (e) the substitutions A39T, N40K, E61V, I66N, P70L, P98G, V99L, I100V, and A101
  • the polypeptide comprises the substitutions C30A, C73A, C93A, and C102A (numbered according to SEQ ID NO:1).
  • the polypeptide is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:320-373.
  • the polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs:320-373.
  • the disclosure also provides a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding any one of the foregoing polypeptides.
  • mRNA messenger RNA
  • ORF open reading frame
  • the ORF is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs:300-317.
  • the ORF comprises the nucleic acid sequence set forth in any one of SEQ ID NOs:300- 317.
  • the mRNA further comprises a 5’ untranslated region (UTR) comprising the nucleic acid sequence of SEQ ID NO:50.
  • the mRNA further comprises a 3’ UTR comprising the nucleic acid sequence of SEQ ID NO:108.
  • the mRNA further comprises a 3’ UTR comprising the nucleic acid sequence of SEQ ID NO:139.
  • the mRNA comprises a 5′ terminal cap.
  • the 5' terminal cap comprises a m 7 GpppG 2 ⁇ OMe , m7G-ppp-Gm-A, m7G-ppp-Gm-AG, Cap0, Cap1, ARCA, inosine, N1-methyl- guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino- guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5′ methylG cap, or an analog thereof.
  • the mRNA comprises a poly-A region.
  • the poly-A region is at least about 10, at least about 20, at Attorney Docket No.45817-0124WO1 / MTX959.20 least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90 nucleotides in length, or at least about 100 nucleotides in length.
  • the poly-A region is at least about 100 nucleotides in length.
  • all of the uracils of the mRNA are N1-methylpseudouracils.
  • the disclosure also provides a pharmaceutical composition comprising any one of the foregoing mRNAs and a pharmaceutically acceptable excipient.
  • the disclosure also provides a lipid nanoparticle comprising any one of the foregoing mRNAs.
  • the disclosure also provides a method of expressing a polypeptide in a human subject in need thereof, the method comprising administering to the human subject an effective amount of any one of the foregoing mRNAs, the foregoing pharmaceutical composition, or the foregoing lipid nanoparticle.
  • the disclosure also provides a method for regulating expression of a polypeptide, the method comprising contacting a cell with: (i) a first polynucleotide comprising (a) a Snu13-binding site, and (b) an open reading frame encoding a first polypeptide; and (ii) a second polynucleotide comprising a nucleotide sequence encoding a second polypeptide, wherein the second polypeptide comprises any one of the foregoing polypeptides; wherein binding of the second polypeptide to the Snu13-binding site represses translation of the first polypeptide from the first polynucleotide.
  • the disclosure also provides a method for regulating expression of a polypeptide in a subject, the method comprising administering to the subject: (i) a first polynucleotide comprising (a) a Snu13-binding site, and (b) an open reading frame encoding a first polypeptide; and (ii) a second polynucleotide comprising a nucleotide sequence encoding a second polypeptide, wherein the second polypeptide comprises any one of the foregoing polypeptides; wherein binding of the Attorney Docket No.45817-0124WO1 / MTX959.20 second polypeptide to the Snu13-binding site represses translation of the first polypeptide from the first polynucleotide.
  • the Snu13-binding site comprises the nucleotide sequence set forth in SEQ ID NO:500.
  • the first polypeptide comprises a secreted protein, a membrane-bound protein, or an intracellular protein.
  • the first polypeptide is a cytokine, an antibody, a vaccine, a receptor, an enzyme, a hormone, a transcription factor, a ligand, a membrane transporter, a structural protein, a nuclease, or a component, variant or fragment thereof.
  • the second polynucleotide is an mRNA.
  • the second polynucleotide comprises any one of the foregoing mRNAs.
  • the first polynucleotide is an mRNA. In some instances, the first polynucleotide is an mRNA in which all of the uracils of the mRNA are N1- methylpseudouracils. [0034] In some instances of the foregoing methods for regulating expression of a polypeptide, the method comprises contacting the cell with, or administering to the subject, the pharmaceutical composition of claim 20 or the lipid nanoparticle of claim 21.
  • the disclosure also provides a composition comprising: (i) a first polynucleotide comprising (a) a Snu13-binding site, and (b) an open reading frame encoding a first polypeptide; and (ii) a second polynucleotide comprising a nucleotide sequence encoding a second polypeptide, wherein the second polypeptide comprises any one of the foregoing polypeptides; wherein binding of the second polypeptide to the Snu13-binding site represses translation of the first polypeptide from the first polynucleotide.
  • the first polynucleotide is an mRNA.
  • the second polynucleotide is an mRNA.
  • the Attorney Docket No.45817-0124WO1 / MTX959.20 first polynucleotide and the second polynucleotide are mRNAs.
  • the Snu13-binding site comprises the nucleotide sequence set forth in SEQ ID NO:500.
  • the first polypeptide comprises a secreted protein, a membrane-bound protein, or an intracellular protein.
  • the first polypeptide is a cytokine, an antibody, a vaccine, a receptor, an enzyme, a hormone, a transcription factor, a ligand, a membrane transporter, a structural protein, a nuclease, or a component, variant or fragment thereof.
  • the first polynucleotide is an mRNA in which all of the uracils of the mRNA are N1- methylpseudouracils.
  • the composition is a lipid nanoparticle.
  • FIG.1A is a cartoon depicting experimental set-up for Snu13-mediated repression of a target mRNA (e.g., encoding enhanced green fluorescent protein (eGFP)).
  • pA polyA tail.
  • FIG.1B is a graph depicting total green intensity area under the curve (AUC) 48 hours after transfection with the indicated mRNA (x-axis) in 10x molar excess of transfected eGFP mRNA containing a Snu13 repressor binding site.
  • Ctrl is mRNA encoding human erythropoietin.
  • FIG.3A is a graph depicting percent expression detected for eGFP with the indicated Snu13 mRNAs.
  • FIG.3B is a graph depicting percent expression detected for NpiLuc with the indicated Snu13 mRNAs.
  • the bars from left to right represent: Snu13 (wt), Snu13_01, Snu13_02, and Snu13_03, respectively.
  • FIG.5A is a graph depicting percent expression detected for eGFP with the indicated mRNAs (x-axis) or mock treatment (cells) at a 20x or 10x molar ratio of G0 eGFP mRNA:G5 NPI-Luc mRNA.
  • FIG.5B is a graph depicting percent expression detected for eGFP with the indicated mRNAs (x-axis) or mock treatment (cells) at a 5x or 1x molar ratio of G0 eGFP mRNA:G5 NPI-Luc mRNA.
  • FIG.5C is a graph depicting percent expression detected for eGFP with the indicated mRNAs (x-axis) or mock treatment (cells) at a 0.2x molar ratio of G0 eGFP mRNA:G5 NPI-Luc mRNA.
  • FIG.6A is a graph depicting percent expression detected for NPI-Luc with the indicated mRNAs (x-axis) or mock treatment (cells) at a 20x or 10x molar ratio of G0 eGFP mRNA:G5 NPI-Luc mRNA.
  • FIG.6B is a graph depicting percent expression detected for NPI-Luc with the indicated mRNAs (x-axis) or mock treatment (cells) at a 5x or 1x molar ratio of G0 eGFP mRNA:G5 NPI-Luc mRNA.
  • FIG.6C is a graph depicting percent expression detected for NPI-Luc with the indicated mRNAs (x-axis) or mock treatment (cells) at a 0.2 molar ratio of G0 eGFP mRNA:G5 NPI-Luc mRNA.
  • DETAILED DESCRIPTION [0052] Efforts to limit off-target effects of RNA-based therapeutics have focused on turning off expression of the therapeutic RNA in undesired cells and locations.
  • the present disclosure is based on the discovery of variant Snu13 polypeptides and polynucleotides.
  • These Snu13 polynucleotides can be used in combination with target polynucleotides comprising Snu13 binding site(s) (e.g., a sequence set forth in any one of SEQ ID NOs: 500-521) and encoding target polypeptides.
  • target polynucleotides comprising Snu13 binding site(s) (e.g., a sequence set forth in any one of SEQ ID NOs: 500-521) and encoding target polypeptides.
  • Snu13 polynucleotides and target polynucleotides can be used in tandem to control the expression of the target polypeptides.
  • the present disclosure provides mRNA therapeutics for regulating expression of a polypeptide (e.g., via regulation of translation of a polynucleotide Attorney Docket No.45817-0124WO1 / MTX959.20 comprising a Snu13 binding site, e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500, and encoding the polypeptide), e.g., for the treatment of various diseases.
  • a polypeptide e.g., via regulation of translation of a polynucleotide Attorney Docket No.45817-0124WO1 / MTX959.20 comprising a Snu13 binding site, e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500, and encoding the polypeptide
  • the Snu13 polynucleotides may be administered in combination (e.g., concurrently or sequentially) with a polynucleotide comprising a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500) and an ORF encoding a polypeptide, such that the encoded Snu13 polypeptide binds to the Snu13-binding site thereby repressing translation of the polypeptide encoded by the ORF.
  • a polynu13-binding site e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500
  • mRNA therapeutics are particularly well-suited for regulating polypeptide expression as the technology provides for the intracellular delivery of first mRNA encoding a Snu13 polypeptide and intracellular delivery of a second mRNA comprising a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500) and an ORF encoding a polypeptide, followed by de novo synthesis of functional Snu13, which regulates expression of the polypeptide encoded by the second mRNA (e.g., binding of the second mRNA to the Snu13 polypeptide represses translation of the second mRNA).
  • a Snu13-binding site e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500
  • an ORF encoding a polypeptide
  • the desired Snu13 protein is expressed by the cells’ own translational machinery, and hence, fully functional Snu13 is expressed.
  • the functional Snu13 may then regulate expression of another polypeptide by, e.g., binding to a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500) in an mRNA encoding the polypeptide.
  • a Snu13-binding site e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500
  • TLRs toll-like receptors
  • ssRNA single-stranded RNA
  • RAG-I retinoic acid-inducible gene I
  • Immune recognition of foreign mRNAs can result in unwanted cytokine effects including interleukin-1 ⁇ (IL-1 ⁇ ) production, tumor necrosis factor- ⁇ (TNF- ⁇ ) distribution and a strong type I interferon (type I IFN) response.
  • IL-1 ⁇ interleukin-1 ⁇
  • TNF- ⁇ tumor necrosis factor- ⁇
  • type I IFN type I interferon
  • This disclosure features the incorporation of different modified nucleotides within mRNAs to minimize the immune activation and optimize the translation Attorney Docket No.45817-0124WO1 / MTX959.20 efficiency of mRNA to protein.
  • Particular aspects feature a combination of nucleotide modification to reduce the innate immune response and sequence optimization, in particular, within the open reading frame (ORF) of mRNAs encoding Snu13 to enhance protein expression.
  • Certain embodiments of the mRNA therapeutic technology of the instant disclosure also feature delivery of mRNA encoding a Snu13 polypeptide described herein (and optionally a target polynucleotide comprising a Snu13 binding site) via a lipid nanoparticle (LNP) delivery system.
  • LNP lipid nanoparticle
  • Lipid nanoparticles are an ideal platform for the safe and effective delivery of mRNAs to target cells.
  • LNPs have the unique ability to deliver nucleic acids by a mechanism involving cellular uptake, intracellular transport and endosomal release or endosomal escape.
  • the instant invention features ionizable lipid-based LNPs combined with mRNA encoding Snu13 (and optionally a target mRNA encoding a target polypeptide and comprising one or more Snu13-binding site(s)) which have improved properties when administered in vivo.
  • the ionizable lipid-based LNP formulations of the invention have improved properties, for example, cellular uptake, intracellular transport and/or endosomal release or endosomal escape.
  • LNPs administered by systemic route e.g., intravenous (IV) administration
  • IV intravenous
  • LNPs administered by systemic route can accelerate the clearance of subsequently injected LNPs, for example, in further administrations.
  • This phenomenon is known as accelerated blood clearance (ABC) and is a key challenge, in particular, when replacing deficient proteins (target proteins) in a therapeutic context. This is because repeat administration of mRNA therapeutics is in most instances essential to maintain necessary levels of target proteins in target tissues in subjects. Repeat dosing challenges can be addressed on multiple levels.
  • mRNA engineering and/or efficient delivery by LNPs can result in increased levels and or enhanced duration of protein being expressed following a first dose of administration, which in turn, can lengthen the time between first dose and subsequent dosing.
  • the ABC phenomenon is, at least in part, transient in nature, with the immune responses underlying ABC resolving after sufficient time following systemic administration.
  • increasing the duration of protein expression and/or activity following systemic delivery of an mRNA therapeutic of the disclosure in one aspect combats the ABC Attorney Docket No.45817-0124WO1 / MTX959.20 phenomenon.
  • LNPs can be engineered to avoid immune sensing and/or recognition and can thus further avoid ABC upon subsequent or repeat dosing.
  • Snu13 is a nuclear protein that is a component of the U4/U6.U5 tri-snRNP and is involved in pre-mRNA splicing. Snu13 binds to the 5’ stem-loop of the U4 snRNA, thereby contributing to spliceosome assembly.
  • CDS coding sequence for wild type SNU13 canonical mRNA sequence, corresponding to transcript variant 2 is described at the NCBI Reference Sequence database (RefSeq) under accession number NM_001003796.1 (“Homo sapiens small nuclear ribonucleoprotein 13 (SNU13), transcript variant 2, mRNA”).
  • the wild type Snu13 canonical protein sequence corresponding to transcript variant 2 is described at the RefSeq database under accession number NP_001003796.1 (“NHP2-like protein 1 [Homo sapiens]”): 1 MTEADVNPKA YPLADAHLTK KLLDLVQQSC NYKQLRKGAN EATKTLNRGI SEFIVMAADA 61 EPLEIILHLP LLCEDKNVPY VFVRSKQALG RACGVSRPVI ACSVTIKEGS QLKQQIQSIQ 121 QSIERLLV (SEQ ID NO:1)
  • the Snu13 protein corresponding to transcript variant 1 is 128 amino acids long.
  • transcript variant 1 of SNU13 is NP_004999.1 and NM_005008.3, respectively.
  • Transcript variant 1 of Snu13 is encoded by the CDS disclosed in the above-mentioned mRNA RefSeq entries.
  • the transcript variant 1 polypeptide is identical to the transcript variant 2 polypeptide (SEQ ID NO:1).
  • the polypeptides of the invention comprise a substitutional variants of a human Snu13 sequence, which can comprise one, two, three or more than three Attorney Docket No.45817-0124WO1 / MTX959.20 substitutions relative to human wild type Snu13 (see, e.g., Table 11, below, for exemplary substitutions in human Snu13).
  • Exemplary Snu13-binding sites are provided in Table 2. See, also, Table 1, below, which provides the amino acid sequence of exemplary Snu13 polypeptides of the invention.
  • a polypeptide of the invention comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence comprises: (i) an amino acid other than leucine (L) at the position corresponding to position 35 of SEQ ID NO:1; Attorney Docket No.45817-0124WO1 / MTX959.20 (ii) an amino acid other than lysine (K) at the position corresponding to position 37 of SEQ ID NO:1; (iii) an amino acid other than alanine (A) at the position corresponding to position 39 of SEQ ID NO:1; (iv) an amino acid other than asparagine (N) at the position corresponding to position 40 of SEQ ID NO:1; (v) an amino acid other than alanine (A) at the position corresponding
  • the polypeptide comprises an alanine (A) at the amino acid corresponding to position 30 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an alanine (A) at the amino acid corresponding to position 73 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an alanine (A) at the amino acid corresponding to position 93 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an alanine (A) at the amino acid corresponding to position 102 of SEQ ID NO:1. In some embodiments, the polypeptide comprises an alanine (A) at each of the amino acids corresponding to positions 30, 73, 93, and 102 of SEQ ID NO:1.
  • the polypeptide comprises a cysteine (C) at the amino acid corresponding to position 30 of SEQ ID NO:1. In some embodiments, the polypeptide comprises a cysteine (C) at the amino acid corresponding to position 73 of SEQ ID NO:1. In some embodiments, the polypeptide comprises a cysteine (C) at the amino acid corresponding to position 93 of SEQ ID NO:1. In some embodiments, the polypeptide comprises a cysteine (C) at the amino acid corresponding to position Attorney Docket No.45817-0124WO1 / MTX959.20 102 of SEQ ID NO:1.
  • the polypeptide comprises a cysteine (C) at each of the amino acids corresponding to positions 30, 73, 93, and 102 of SEQ ID NO:1.
  • the polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:320-373.
  • the polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs:320-373.
  • the polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs:320-337.
  • the polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs:338-355. In some embodiments, the polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs:356-373.
  • a polypeptide of the invention comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence comprises: (i) an amino acid other than alanine (A) at the position corresponding to position 39 of SEQ ID NO:1, (ii) an amino acid other than asparagine (N) at the position corresponding to position 40 of SEQ ID NO:1, (iii) an amino acid other than glutamic acid (E) at the position corresponding to position 61 of SEQ ID NO:1, (iv) an amino acid other than isoleucine (I) at the position corresponding to position 65 of SEQ ID NO:1, (v) an amino acid other than lysine (K) at the position
  • the polypeptide comprises: one or more substitutions selected from the group consisting of A39N, N40Y, E61S, I65K, K86S, and Q114H (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises: the substitutions A39N, N40Y, E61S, I65K, K86S, and Q114H (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises the substitutions C30A, C73A, C93A, and C102A (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises an alanine (A) at each of the amino acids corresponding to positions 30, 73, 93, and 102 of SEQ ID NO:1.
  • the polypeptide has at least 90%, at least Attorney Docket No.45817-0124WO1 / MTX959.20 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:320, 338, and 356.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:320.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:338.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:356.
  • a polypeptide of the invention comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence comprises: (i) an amino acid other than lysine (K) at the position corresponding to position 37 of SEQ ID NO:1; (ii) an amino acid other than alanine (A) at the position corresponding to position 39 of SEQ ID NO:1; (iii) an amino acid other than asparagine (N) at the position corresponding to position 40 of SEQ ID NO:1; (iv) an amino acid other than glutamic acid (E) at the position corresponding to position 61 of SEQ ID NO:1; (v) an amino acid other than proline (P) at the position corresponding to position 62 of SEQ ID NO:1; (vi) an amino acid other than isoleucine (I) at the position corresponding to position 65 of SEQ ID NO:1; (vii) an amino acid other than isoleucine (I) at the position corresponding to position
  • the polypeptide comprises: (b) one or more substitutions selected from the group consisting of K37R, A39V, N40H, E61H, P62H, I65K, I66L, and K86S (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises: the substitutions K37R, A39V, N40H, E61H, P62H, I65K, I66L, and K86S (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises the substitutions C30A, C73A, C93A, and C102A (numbered according to SEQ ID NO:1).
  • the polypeptide comprises an alanine (A) at each of the amino acids corresponding to positions 30, 73, 93, and 102 of SEQ ID NO:1.
  • the polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:321, 339, and 357.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:321.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:339. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:357.
  • a polypeptide of the invention comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence comprises: (i) an amino acid other than lysine (K) at the position corresponding to position 37 of SEQ ID NO:1; (ii) an amino acid other than alanine (A) at the position corresponding to position 39 of SEQ ID NO:1; (iii) an amino acid other than asparagine (N) at the position corresponding to position 40 of SEQ ID NO:1; (iv) an amino acid other than isoleucine (I) at the position corresponding to position 65 of SEQ ID NO:1; (v) an amino acid other than isoleucine (I) at the position corresponding to position 66 of SEQ
  • the polypeptide comprises: one or more substitutions selected from the group consisting of K37T, A39R, N40H, I65K, I66V, and K86S (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises: the substitutions K37T, A39R, N40H, I65K, I66V, and K86S (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises the substitutions C30A, C73A, C93A, and C102A (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises an alanine (A) at each of the amino acids corresponding to positions 30, 73, 93, and 102 of SEQ ID NO:1.
  • the polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:322, 340, and 358.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:322.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:340.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:358.
  • a polypeptide of the invention comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence comprises: (i) an amino acid other than alanine (A) at the position corresponding to position 39 of SEQ ID NO:1; (ii) an amino acid other than asparagine (N) at the position corresponding to position 40 of SEQ ID NO:1; (iii) an amino acid other than alanine (A) at the position corresponding to position 42 of SEQ ID NO:1; (iv) an amino acid other than alanine (A) at the position corresponding to position 60 of SEQ ID NO:1; (v) an amino acid other than glutamic acid (E) at the position corresponding to position 61 of SEQ ID NO:1; (vi) an amino acid other than isoleucine (I) at the position corresponding to position 65 of
  • the polypeptide comprises: one or more substitutions selected from the group consisting of A39V, N40K, A42V, A60V, E61S, I65V, V95I, S96D, R97M, V99L, A101V, K113S, and Q114N (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises: the substitutions A39V, N40K, A42V, A60V, E61S, I65V, V95I, S96D, R97M, V99L, A101V, K113S, and Q114N (numbered according to SEQ ID NO:1).
  • the polypeptide comprises the substitutions C30A, C73A, C93A, and C102A (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises an alanine (A) at each of the amino acids corresponding to positions 30, 73, 93, and 102 of SEQ ID NO:1. In some embodiments, the polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of Attorney Docket No.45817-0124WO1 / MTX959.20 SEQ ID NOs:323, 341, and 359.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:323. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:341. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:359.
  • a polypeptide of the invention comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence comprises: (i) an amino acid other than alanine (A) at the position corresponding to position 39 of SEQ ID NO:1; (ii) an amino acid other than asparagine (N) at the position corresponding to position 40 of SEQ ID NO:1; (iii) an amino acid other than glutamic acid (E) at the position corresponding to position 61 of SEQ ID NO:1; (iv) an amino acid other than isoleucine (I) at the position corresponding to position 66 of SEQ ID NO:1; (v) an amino acid other than proline (P) at the position corresponding to position 70 of SEQ ID NO:1; (vi) an amino acid other than proline (P) at the position corresponding to position 98 of SEQ ID NO:1; (vii) an amino acid other than valine (V) at the position
  • the polypeptide comprises: one or more substitutions selected from the group consisting of A39T, N40K, E61V, I66N, P70L, P98G, V99L, I100V, and A101V (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises: the substitutions A39T, N40K, E61V, I66N, P70L, P98G, V99L, I100V, and A101V (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises the substitutions C30A, C73A, C93A, and C102A (numbered according to SEQ ID NO:1).
  • the polypeptide comprises an alanine (A) at each of the amino acids corresponding to positions 30, 73, 93, and 102 of SEQ ID NO:1.
  • the polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:324, 342, and 360.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:324.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:342. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:360.
  • a polypeptide of the invention comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence comprises: (i) an amino acid other than leucine (L) at the position corresponding to position 35 of SEQ ID NO:1; (ii) an amino acid other than lysine (K) at the position corresponding to position 37 of SEQ ID NO:1; (iii) an amino acid other than alanine (A) at the position corresponding to position 39 of SEQ ID NO:1; (iv) an amino acid other than alanine (A) at the position corresponding to position 42 of SEQ ID NO:1; (v) an amino acid other than isoleucine (I) at the position corresponding to position 65 of SEQ ID NO:1
  • the polypeptide comprises: one or more substitutions selected from the group consisting of L35I, K37T, A39V, A42V, I65V, V95L, S96E, V99M, and A101V (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises: the substitutions L35I, K37T, A39V, A42V, I65V, V95L, S96E, V99M, and A101V (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises the substitutions C30A, C73A, C93A, and C102A (numbered according to SEQ ID NO:1).
  • the polypeptide comprises an alanine (A) at each of the amino acids corresponding to positions 30, 73, 93, and 102 of SEQ ID NO:1.
  • the polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:325, 343, and 361.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:325.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:343. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:361.
  • a polypeptide of the invention comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence comprises: (i) an amino acid other than lysine (K) at the position corresponding to position 37 of SEQ ID NO:1; (ii) an amino acid other than alanine (A) at the position corresponding to position 39 of SEQ ID NO:1; (iii) an amino acid other than asparagine (N) at the position corresponding to position 40 of SEQ ID NO:1; (iv) an amino acid other than alanine (A) at the position corresponding to position 60 of SEQ ID NO:1; (v) an amino acid other than isoleucine (I) at the position corresponding to position 65 of SEQ ID NO:1; (vi) an amino acid other than valine (V) at the position corresponding to position 95 of SEQ ID NO:1; (vii) an amino acid other than serine (S) at the position
  • the polypeptide comprises: one or more substitutions selected from the group consisting of K37R, A39V, N40K, A60M, I65V, V95I, S96D, R97G, P98A, V99M, I100A, and A101V (numbered according to SEQ ID NO:1).
  • the polypeptide comprises: the substitutions K37R, A39V, N40K, A60M, I65V, V95I, S96D, R97G, P98A, V99M, I100A, and A101V (numbered according to SEQ ID NO:1).
  • the polypeptide comprises the substitutions C30A, C73A, C93A, and C102A (numbered according to SEQ ID NO:1).
  • the polypeptide comprises an alanine (A) at each of the amino acids corresponding to positions 30, 73, 93, and 102 of SEQ ID NO:1.
  • the polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, Attorney Docket No.45817-0124WO1 / MTX959.20 at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:326, 344, and 362.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:326.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:344. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:362. [0072] in some embodiments, a polypeptide of the invention comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence comprises: (i) an amino acid other than leucine (L) at the position corresponding to position 35 of SEQ ID NO:1; (ii) an amino acid other than lysine (K) at the position corresponding to position 37 of SEQ ID NO:1; (iii) an amino acid other than alanine (A) at the position corresponding to position 39 of SEQ ID NO:1; (iv) an amino acid other than asparagine (N) at the position corresponding to position 40 of SEQ ID NO:1; (v) an amino acid other than alanine (A) at the position corresponding to position 42 of SEQ ID NO:1;
  • the polypeptide comprises: one or more substitutions selected from the group consisting of L35I, K37T, A39T, N40K, A42V, A60T, E61S, I65V, K86Q, V95I, S96K, V99L, I100T, and A101V (numbered according to SEQ ID NO:1).
  • the polypeptide comprises: the substitutions L35I, K37T, A39T, N40K, A42V, A60T, E61S, I65V, K86Q, V95I, Attorney Docket No.45817-0124WO1 / MTX959.20 S96K, V99L, I100T, and A101V (numbered according to SEQ ID NO:1).
  • the polypeptide comprises the substitutions C30A, C73A, C93A, and C102A (numbered according to SEQ ID NO:1).
  • the polypeptide comprises an alanine (A) at each of the amino acids corresponding to positions 30, 73, 93, and 102 of SEQ ID NO:1.
  • the polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:327, 345, and 363.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:327. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:345. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:363.
  • a polypeptide of the invention comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence comprises: (i) an amino acid other than asparagine (N) at the position corresponding to position 40 of SEQ ID NO:1; (ii) an amino acid other than alanine (A) at the position corresponding to position 42 of SEQ ID NO:1; (iii) an amino acid other than alanine (A) at the position corresponding to position 60 of SEQ ID NO:1; (iv) an amino acid other than isoleucine (I) at the position corresponding to position 65 of SEQ ID NO:1; (v) an amino acid other than lysine (K) at the position corresponding to position 86 of SEQ ID NO:1; (vi) an amino acid other than valine (V) at the position corresponding to position 95 of SEQ ID NO:1; (vii) an amino acid other than serine (S) at the position
  • the polypeptide comprises: one or more substitutions selected from the group consisting of N40K, A42V, A60M, I65V, K86Q, V95L, S96D, R97V, I100A, A101V, and Q114H (numbered according to SEQ Attorney Docket No.45817-0124WO1 / MTX959.20 ID NO:1).
  • the polypeptide comprises: the substitutions N40K, A42V, A60M, I65V, K86Q, V95L, S96D, R97V, I100A, A101V, and Q114H (numbered according to SEQ ID NO:1).
  • the polypeptide comprises the substitutions C30A, C73A, C93A, and C102A (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises an alanine (A) at each of the amino acids corresponding to positions 30, 73, 93, and 102 of SEQ ID NO:1. In some embodiments, the polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:328, 346, and 364.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:328. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:346. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:364.
  • a polypeptide of the invention comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence comprises: (i) an amino acid other than alanine (A) at the position corresponding to position 39 of SEQ ID NO:1; (ii) an amino acid other than asparagine (N) at the position corresponding to position 40 of SEQ ID NO:1; (iii) an amino acid other than alanine (A) at the position corresponding to position 60 of SEQ ID NO:1; (iv) an amino acid other than glutamic acid (E) at the position corresponding to position 61 of SEQ ID NO:1; (v) an amino acid other than isoleucine (I) at the position corresponding to position 65 of SEQ ID NO:1; (vi) an amino acid other than lysine (K) at the position corresponding to position 86 of SEQ ID NO:1; (vii) an amino acid other than proline (P)
  • the polypeptide comprises: one or more substitutions selected from the group consisting of A39V, N40K, A60T, E61T, I65V, K86Q, P98G, I100F, and S110F (numbered according to SEQ ID NO:1).
  • the polypeptide Attorney Docket No.45817-0124WO1 / MTX959.20 comprises: the substitutions A39V, N40K, A60T, E61T, I65V, K86Q, P98G, I100F, and S110F (numbered according to SEQ ID NO:1).
  • the polypeptide comprises the substitutions C30A, C73A, C93A, and C102A (numbered according to SEQ ID NO:1).
  • the polypeptide comprises an alanine (A) at each of the amino acids corresponding to positions 30, 73, 93, and 102 of SEQ ID NO:1.
  • the polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:329, 347, and 365.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:329.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:347.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:365.
  • a polypeptide of the invention comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence comprises: (i) an amino acid other than alanine (A) at the position corresponding to position 39 of SEQ ID NO:1; (ii) an amino acid other than asparagine (N) at the position corresponding to position 40 of SEQ ID NO:1; (iii) an amino acid other than glutamic acid (E) at the position corresponding to position 61 of SEQ ID NO:1; (iv) an amino acid other than isoleucine (I) at the position corresponding to position 65 of SEQ ID NO:1; (v) an amino acid other than lysine (K) at the position corresponding to position 86 of SEQ ID NO:1; (vi) an amino acid other than lysine (K) at the position corresponding
  • the polypeptide comprises: one or more substitutions selected from the group consisting of A39N, N40Y, E61S, I65K, K86S, K113S, and Q114N (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises: the substitutions A39N, N40Y, E61S, I65K, K86S, K113S, and Q114N (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises the substitutions C30A, C73A, C93A, and C102A (numbered according to SEQ ID NO:1).
  • the Attorney Docket No.45817-0124WO1 / MTX959.20 polypeptide comprises an alanine (A) at each of the amino acids corresponding to positions 30, 73, 93, and 102 of SEQ ID NO:1.
  • the polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:330, 348, and 366.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:330.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:348. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:366. [0076] In some embodiments, a polypeptide of the invention comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence comprises: (i) an amino acid other than alanine (A) at the position corresponding to position 39 of SEQ ID NO:1; (ii) an amino acid other than asparagine (N) at the position corresponding to position 40 of SEQ ID NO:1; (iii) an amino acid other than glutamic acid (E) at the position corresponding to position 61 of SEQ ID NO:1; (iv) an amino acid other than proline (P) at the position corresponding to position 62 of SEQ ID NO:1; (v) an amino acid other than isoleucine (I) at the position corresponding to position 65 of SEQ ID NO:1
  • the polypeptide comprises: one or more substitutions selected from the group consisting of A39V, N40H, E61H, P62H, I65K, I66L, K86S, K113S, and Q114N (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises: the substitutions A39V, N40H, E61H, P62H, I65K, I66L, K86S, K113S, and Q114N (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises the substitutions C30A, C73A, C93A, and C102A (numbered according to SEQ ID NO:1).
  • the polypeptide comprises an alanine (A) at each of the amino acids corresponding to Attorney Docket No.45817-0124WO1 / MTX959.20 positions 30, 73, 93, and 102 of SEQ ID NO:1.
  • the polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:331, 349, and 367.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:331.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:349. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:367. [0077] In some embodiments, a polypeptide of the invention comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence comprises: (i) an amino acid other than alanine (A) at the position corresponding to position 39 of SEQ ID NO:1; (ii) an amino acid other than asparagine (N) at the position corresponding to position 40 of SEQ ID NO:1; (iii) an amino acid other than glutamic acid (E) at the position corresponding to position 61 of SEQ ID NO:1; (iv) an amino acid other than isoleucine (I) at the position corresponding to position 65 of SEQ ID NO:1; (v) an amino acid other than isoleucine (I) at the position corresponding to position 66 of S
  • the polypeptide comprises: one or more substitutions selected from the group consisting of A39I, N40Y, E61K, I65Q, I66A, K86S, K113S, and Q114N (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises: the substitutions A39I, N40Y, E61K, I65Q, I66A, K86S, K113S, and Q114N (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises the substitutions C30A, C73A, C93A, and C102A (numbered according to SEQ ID NO:1).
  • the polypeptide comprises an alanine (A) at each of the amino acids corresponding to positions 30, 73, 93, and 102 of SEQ ID NO:1.
  • the polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, Attorney Docket No.45817-0124WO1 / MTX959.20 at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:332, 350, and 368.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:332.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:350. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:368. [0078] In some embodiments, a polypeptide of the invention comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence comprises: (i) an amino acid other than alanine (A) at the position corresponding to position 39 of SEQ ID NO:1; (ii) an amino acid other than asparagine (N) at the position corresponding to position 40 of SEQ ID NO:1; (iii) an amino acid other than glutamic acid (E) at the position corresponding to position 61 of SEQ ID NO:1; (iv) an amino acid other than isoleucine (I) at the position corresponding to position 65 of SEQ ID NO:1; (v) an amino acid other than isoleucine (I) at the position corresponding to position 66 of S
  • the polypeptide comprises: one or more substitutions selected from the group consisting of A39N, N40Y, E61S, I65K, I66L, and K86S (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises: the substitutions A39N, N40Y, E61S, I65K, I66L, and K86S (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises the substitutions C30A, C73A, C93A, and C102A (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises an alanine (A) at each of the amino acids corresponding to positions 30, 73, 93, and 102 of SEQ ID NO:1.
  • the polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:333, 351, and 369.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:333.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:351.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:369.
  • a polypeptide of the invention comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence comprises: (i) an amino acid other than alanine (A) at the position corresponding to position 39 of SEQ ID NO:1; (ii) an amino acid other than asparagine (N) at the position corresponding to position 40 of SEQ ID NO:1; (iii) an amino acid other than proline (P) at the position corresponding to position 62 of SEQ ID NO:1; (iv) an amino acid other than isoleucine (I) at the position corresponding to position 65 of SEQ ID NO:1; (v) an amino acid other than isoleucine (I) at the position corresponding to position 66 of SEQ ID NO:1; (vi) an amino acid other than lysine (K) at the position corresponding to position 86 of SEQ ID NO:1; (vii) an amino acid other than proline (P)
  • the polypeptide comprises: one or more substitutions selected from the group consisting of A39T, N40Y, P62H, I65K, I66L, K86S, P98S, K113S, and Q114N (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises: the substitutions A39T, N40Y, P62H, I65K, I66L, K86S, P98S, K113S, and Q114N (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises the substitutions C30A, C73A, C93A, and C102A (numbered according to SEQ ID NO:1).
  • the polypeptide comprises an alanine (A) at each of the amino acids corresponding to positions 30, 73, 93, and 102 of SEQ ID NO:1.
  • the polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:334, 352, and 370.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:334.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID Attorney Docket No.45817-0124WO1 / MTX959.20 NO:352. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:370.
  • a polypeptide of the invention comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence comprises: (i) an amino acid other than alanine (A) at the position corresponding to position 39 of SEQ ID NO:1; (ii) an amino acid other than asparagine (N) at the position corresponding to position 40 of SEQ ID NO:1; (iii) an amino acid other than isoleucine (I) at the position corresponding to position 65 of SEQ ID NO:1; (iv) an amino acid other than isoleucine (I) at the position corresponding to position 66 of SEQ ID NO:1; (v) an amino acid other than lysine (K) at the position corresponding to position 86 of SEQ ID NO:1; (vi) an amino acid other than lysine (K) at the position corresponding to position 113 of SEQ ID NO:1; and (vii) an amino acid other than glutamine
  • the polypeptide comprises: one or more substitutions selected from the group consisting of A39Y, N40Y, I65K, I66M, K86S, K113S, and Q114N (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises: the substitutions A39Y, N40Y, I65K, I66M, K86S, K113S, and Q114N (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises the substitutions C30A, C73A, C93A, and C102A (numbered according to SEQ ID NO:1).
  • the polypeptide comprises an alanine (A) at each of the amino acids corresponding to positions 30, 73, 93, and 102 of SEQ ID NO:1.
  • the polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:335, 353, and 371.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:335.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:353.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:371.
  • a polypeptide of the invention comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID Attorney Docket No.45817-0124WO1 / MTX959.20 NO:1, wherein the amino acid sequence comprises: (i) an amino acid other than alanine (A) at the position corresponding to position 39 of SEQ ID NO:1; (ii) an amino acid other than asparagine (N) at the position corresponding to position 40 of SEQ ID NO:1; (iii) an amino acid other than aspartic acid (D) at the position corresponding to position 59 of SEQ ID NO:1; (iv) an amino acid other than proline (P) at the position corresponding to position 62 of SEQ ID NO:1; (v) an amino acid other than isoleucine (I) at the position corresponding to position 65 of SEQ ID NO:1; (
  • the polypeptide comprises: one or more substitutions selected from the group consisting of A39T, N40H, D59N, P62H, I65K, I66L, K86S, and L112F (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises: the substitutions A39T, N40H, D59N, P62H, I65K, I66L, K86S, and L112F (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises the substitutions C30A, C73A, C93A, and C102A (numbered according to SEQ ID NO:1).
  • the polypeptide comprises an alanine (A) at each of the amino acids corresponding to positions 30, 73, 93, and 102 of SEQ ID NO:1.
  • the polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:336, 354, and 372.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:336.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:354.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:372.
  • a polypeptide of the invention comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence comprises: (i) an amino acid other than alanine (A) at the position corresponding to position 39 of SEQ ID NO:1; (ii) an Attorney Docket No.45817-0124WO1 / MTX959.20 amino acid other than asparagine (N) at the position corresponding to position 40 of SEQ ID NO:1; (iii) an amino acid other than glutamic acid (E) at the position corresponding to position 61 of SEQ ID NO:1; (iv) an amino acid other than proline (P) at the position corresponding to position 62 of SEQ ID NO:1; (v) an amino acid other than isoleucine (I) at the position corresponding to position 65 of SEQ ID NO:1; (
  • the polypeptide comprises: one or more substitutions selected from the group consisting of A39T, N40K, E61K, P62H, I65R, I66M, K86S, K113S, and Q114N (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises: the substitutions A39T, N40K, E61K, P62H, I65R, I66M, K86S, K113S, and Q114N (numbered according to SEQ ID NO:1). In some embodiments, the polypeptide comprises the substitutions C30A, C73A, C93A, and C102A (numbered according to SEQ ID NO:1).
  • the polypeptide comprises an alanine (A) at each of the amino acids corresponding to positions 30, 73, 93, and 102 of SEQ ID NO:1.
  • the polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:337, 355, and 373.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:337.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:355.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:373.
  • the Snu13 polypeptides of the invention bind to and repress a target RNA.
  • the target RNA comprises a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500) and an ORF encoding a polypeptide.
  • the target RNA comprises the sequence of SEQ ID NO:500 and an ORF encoding a polypeptide.
  • the Snu13 polypeptide binds to a second polynucleotide, wherein the second polynucleotide comprises a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500) and an ORF encoding a second polypeptide.
  • a Snu13-binding site e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500
  • the Snu13 polypeptide binds to the second polynucleotide and represses translation of the second polynucleotide by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% (as compared to translation of the second polynucleotide in the absence of the Snu13 polypeptide).
  • the Snu13 polypeptide binds to the second polynucleotide and represses translation of the second polynucleotide by 5-20%, 5-30%, 5-40%, 5-50%, 10-4%, 10-50%, 10-60%, 25-75%, 25-85%, 25-95%, 50-75%, 50-85%, or 75-95% (as compared to translation of the second polynucleotide in the absence of the Snu13 polypeptide).
  • Methods of assessing the translation of a polynucleotide are known in the art and described in the working examples herein, e.g., reporter assays, western blot, immunofluorescence, FACS, and ELISA.
  • the Snu13 polypeptide has a Snu13 activity which is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of the Snu13 activity of the corresponding wild-type Snu13 protein (i.e., the same Snu13 protein but without the mutation(s)).
  • Methods for determining Snu13 activity are known in the art; see, e.g., the working examples described herein.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprising an ORF encoding a Snu13 polypeptide is sequence optimized.
  • the Snu13 polypeptide of the invention comprises one or more additional mutations (relative to SEQ ID NO:1, e.g., one or more mutations other than those described in Table 11) that do not alter Snu13 protein activity. Such mutant Snu13 polypeptides can be referred to as function-neutral.
  • the polynucleotide comprises an ORF that encodes a mutant Snu13 polypeptide comprising one or more function-neutral point mutations.
  • a Snu13 polypeptide described herein has higher Snu13 protein activity than the corresponding wild-type Snu13 protein.
  • the Snu13 polypeptide has a Snu13 activity that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% higher than the activity of the corresponding wild-type Snu13 protein (i.e., the same Snu13 protein but without the mutation(s)).
  • a Snu13 polypeptide described herein is selective for G5-modified mRNA (compared to unmodified mRNA).
  • the Snu13 polypeptide has a Snu13 activity against G5-modified mRNA that is at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.25-fold, at least 2.5-fold, at least 2.75-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold higher than the Snu13 activity against unmodified (G0) mRNA (i.e., mRNA in which none of the uracils are modified with G5).
  • G0 unmodified
  • the disclosure provides a polynucleotide (e.g., a RNA, e.g., a mRNA) comprising a nucleotide sequence (e.g., an open reading frame (ORF)) encoding a Snu13 polypeptide described herein.
  • a polynucleotide e.g., a RNA, e.g., a mRNA
  • a nucleotide sequence e.g., an open reading frame (ORF)
  • ORF open reading frame
  • sequence tags or amino acids can be added to the sequences encoded by the polynucleotides of the invention (e.g., at the N-terminal or C-terminal ends), e.g., for localization.
  • amino acid residues located at the carboxy, amino terminal, or internal regions of a polypeptide of the invention can optionally be deleted providing for fragments.
  • Snu13 protein fragments, functional protein domains, variants, and homologous proteins (orthologs) are also within the scope of the Snu13 polypeptides of the disclosure.
  • Nonlimiting examples of polypeptides encoded by the Snu13 polynucleotides of the invention are set forth in SEQ ID NOs:320-373. Attorney Docket No.45817-0124WO1 / MTX959.20 2.
  • the instant invention features polynucleotides (e.g., a RNA, e.g., an mRNA) encoding Snu13 polypeptides.
  • polynucleotides e.g., a RNA, e.g., an mRNA
  • the polynucleotides encoding Snu13 polypeptides may be used in combination with a second polynucleotide (e.g., a RNA, e.g., an mRNA) encoding a second polypeptide (e.g., a target polypeptide), wherein the second polynucleotide comprises a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500).
  • a second polynucleotide e.g., a RNA, e.g., an mRNA
  • a second polypeptide e.g., a target polypeptide
  • the second polynucleotide comprises a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500).
  • the target polypeptide comprises a secreted protein, a membrane-bound protein, or an intracellular protein.
  • the target polypeptide is a cytokine, an antibody, a vaccine, a receptor, an enzyme, a hormone, a transcription factor, a ligand, a membrane transporter, a structural protein, a nuclease, or a component, variant or fragment thereof.
  • the polynucleotides e.g., RNAs, e.g., mRNAs
  • the invention relates, in part, to polynucleotides, e.g., mRNAs, comprising an open reading frame of linked nucleosides encoding a Snu13 polypeptide described herein, isoforms thereof, variants thereof, functional fragments thereof, and fusion proteins comprising the Snu13 polypeptide.
  • the invention provides sequence-optimized polynucleotides comprising nucleotides encoding a Snu13 polypeptide described herein, or sequence having high sequence identity with those sequence optimized polynucleotides.
  • the invention provides polynucleotides (e.g., RNAs, e.g., mRNAs) that comprise a nucleotide sequence (e.g., an ORF) encoding one or more (e.g., 1, 2, 3 or more) Snu13 polypeptides described herein.
  • the encoded Snu13 polypeptide of the invention can be selected from: Attorney Docket No.45817-0124WO1 / MTX959.20 (i) a full length Snu13 polypeptide described herein (e.g., any one of SEQ ID NOs:320-373 or having one or more of the substitutions described in Table 11); (ii) a functional fragment of a Snu13 polypeptide described herein (e.g., a truncated (e.g., deletion of carboxy, amino terminal, or internal regions) sequence shorter than Snu13; but still retaining Snu13 activity); (iii) a variant thereof (e.g., full length or truncated Snu13 polypeptides in which one or more amino acids have been replaced, e.g., variants that retain all or most of the Snu13 activity of the polypeptide with respect to a reference protein (e.g., any natural or artificial variants known in the art));
  • the polynucleotide e.g., a RNA, e.g., an mRNA
  • the polynucleotide is introduced to the cells in vitro. In some embodiments, the polynucleotide is introduced to the cells in vivo. [0097] In some embodiments, the polynucleotides (e.g., a RNA, e.g., an mRNA) of the invention comprise a nucleotide sequence (e.g., an ORF) that encodes a Snu13 polypeptide, e.g., any one of SEQ ID NOs:320-373.
  • a nucleotide sequence e.g., an ORF
  • the polynucleotides e.g., a RNA, e.g., an mRNA
  • the polynucleotides of the invention comprise a nucleotide sequence (e.g., an ORF) that encodes a variant of a Snu13 polypeptide described herein, e.g., a variant of any one of SEQ ID Attorney Docket No.45817-0124WO1 / MTX959.20 NOs:320-373.
  • the variant of any one of SEQ ID NOs:320-373 retains the substitutions present in any one of SEQ ID NOs:320-373, respectively, relative to wild type Snu13.
  • the polynucleotides (e.g., a RNA, e.g., an mRNA) of the invention comprise a nucleotide sequence (e.g., an ORF) that encodes a fragment of a Snu13 polypeptide described herein.
  • the polynucleotides (e.g., a RNA, e.g., an mRNA) of the invention comprise a nucleotide sequence (e.g., an ORF) that encodes a Snu13 fusion protein.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a codon optimized nucleic acid sequence, wherein the open reading frame (ORF) of the codon optimized nucleic acid sequence is derived from a wild type Snu13 protein sequence (e.g., wild type human Snu13).
  • ORF open reading frame
  • the corresponding wild type sequence is the native human Snu13.
  • the corresponding wild type sequence is the corresponding fragment from the wild-type human Snu13.
  • the polynucleotides (e.g., a RNA, e.g., an mRNA) of the invention comprise a nucleotide sequence (e.g., an ORF) encoding a functional fragment of a Snu13 polypeptide described herein, wherein the functional fragment retains Snu13 protein activity.
  • the Snu13 protein fragment has activity which is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of the Snu13 protein activity of the corresponding full length Snu13 protein.
  • the polynucleotides e.g., a RNA, e.g., an mRNA
  • the polynucleotides comprising an ORF encoding a functional Snu13 protein fragment is sequence optimized.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding a Snu13 protein fragment that has higher Snu13 protein activity, respectively, than the Attorney Docket No.45817-0124WO1 / MTX959.20 corresponding full length Snu13 protein.
  • a nucleotide sequence e.g., an ORF
  • the Snu13 protein fragment has Snu13 activity that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% higher than the Snu13 activity of the corresponding full length Snu13 protein.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding a Snu13 protein fragment that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25% shorter than wild-type Snu13 protein, respectively.
  • a nucleotide sequence e.g., an ORF
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding a Snu13 polypeptide (e.g., a variant Snu13 polypeptide described herein or a functional fragment or variant thereof), wherein the nucleotide sequence is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of any one of SEQ ID NOs:300-317.
  • a nucleotide sequence e.g., an ORF
  • Snu13 polypeptide e.g., a variant Snu13 polypeptide described herein or a functional fragment or variant thereof
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding a Snu13 polypeptide (e.g., a variant Snu13 polypeptide described herein or a functional fragment thereof), wherein the nucleotide sequence has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least at least
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding a Snu13 polypeptide (e.g., a variant Snu13 polypeptide described herein or a functional fragment thereof), wherein the nucleotide sequence has 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 70% to 95%, 80% to 95%, 70% to 85%, 75% to 90%, Attorney Docket No.45817-0124WO1 / MTX959.20 80% to 95%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100%, sequence identity to the sequence of any one of SEQ ID NOs:300-317.
  • a nucleotide sequence e.g., an ORF
  • a Snu13 polypeptide e.g.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding a Snu13 polypeptide (e.g., a variant Snu13 polypeptide described herein or a functional fragment thereof), wherein the nucleotide sequence is between 70% and 90% identical; between 75% and 85% identical; between 76% and 84% identical; between 77% and 83% identical, between 77% and 82% identical, or between 78% and 81% identical to the sequence of any one of SEQ ID NOs:300-317.
  • a nucleotide sequence e.g., an ORF
  • a Snu13 polypeptide e.g., a variant Snu13 polypeptide described herein or a functional fragment thereof
  • the nucleotide sequence is between 70% and 90% identical; between 75% and 85% identical; between 76% and 84% identical; between 77% and
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises from about 300 to about 100,000 nucleotides (e.g., from 300 to 1,000, from 300 to 2,000, from 300 to 3,000, from 300 to 4,000, from 300 to 4,500, from 300 to 5,000, from 300 to 5,500, 350 to 2,000, from 350 to 3,000, from 350 to 4,000, from 350 to 4,500, from 350 to 5,000, from 350 to 5,500, 390 to 3,000, from 390 to 4,000, from 390 to 4,500, from 390 to 5,000, from 390 to 5,500, from 500 to 4,000, from 500 to 4,500, from 500 to 5,000, from 500 to 5,500, from 1,000 to 4,500, from 1,000 to 5,000, from 1,000 to 5,500, 1,500 to 5,000, from 1,500 to 5,500, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000
  • the polynucleotide of the invention (e.g., a RNA, e.g., an mRNA) comprises a nucleotide sequence (e.g., an ORF) encoding a Snu13 polypeptide (e.g., a variant Snu13 polypeptide described herein or a functional fragment thereof), wherein the length of the nucleotide sequence (e.g., an ORF) is at least 390 nucleotides in length (e.g., at least or greater than about 390, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,900, 4,000, 4,100
  • the polynucleotide of the invention (e.g., a RNA, e.g., an mRNA) comprises a nucleotide sequence (e.g., an ORF, e.g., any one of SEQ ID NOs:300-317) encoding a Snu13 polypeptide (e.g., a variant Snu13 polypeptide described herein or a functional fragment thereof) and further comprises at least one nucleic acid sequence that is noncoding, e.g., a microRNA binding site.
  • a nucleotide sequence e.g., an ORF, e.g., any one of SEQ ID NOs:300-317
  • Snu13 polypeptide e.g., a variant Snu13 polypeptide described herein or a functional fragment thereof
  • at least one nucleic acid sequence that is noncoding, e.g., a microRNA binding site.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention further comprises a 5′ UTR (e.g., set forth in Table 4, e.g., SEQ ID NO:50) and a 3′ UTR (e.g., set forth in Table 5 or Table 7, e.g., SEQ ID NO:108).
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises the sequence of any one of SEQ ID NOs:300-317.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a 5′ terminal cap (e.g., m7Gp-ppGm-A, Cap0, Cap1, ARCA, inosine, N1-methyl- guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino- guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5′ methylG cap, or an analog thereof) and a poly-A-tail region (e.g., about 100 nucleotides in length).
  • a 5′ terminal cap e.g., m7Gp-ppGm-A, Cap0, Cap1, ARCA, inosine, N1-methyl- guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a 5′ UTR comprising a nucleic acid sequence of SEQ ID NO:50.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a 3′ UTR comprising a nucleic acid sequence of SEQ ID NO:108.
  • the mRNA comprises a polyA tail.
  • the poly A tail is 50-150 (SEQ ID NO:197), 75-150 (SEQ ID NO:198), 85-150 (SEQ ID NO:199), 90-120 (SEQ ID NO:193), 90-130 (SEQ ID NO:194), or 90-150 (SEQ ID NO:192) nucleotides in length. In some instances, the poly A tail is 100 nucleotides in length (SEQ ID NO:195). In some instances, the poly A tail is protected (e.g., with an inverted deoxy- thymidine). In some instances, the poly A tail comprises A100-UCUAG-A20- inverted deoxy-thymidine (SEQ ID NO:211).
  • the poly A tail is A100-UCUAG-A20-inverted deoxy-thymidine (SEQ ID NO:211).
  • the polynucleotide of the invention e.g., a RNA, e.g., an mRNA
  • a polynucleotide of the invention comprising a nucleotide sequence (e.g., an ORF) encoding a Snu13 polypeptide is DNA or RNA.
  • the polynucleotide of the invention is RNA.
  • the polynucleotide of the invention is, or functions as, an mRNA.
  • the mRNA comprises a nucleotide sequence (e.g., an ORF) that encodes at least one Snu13 polypeptide, and is capable of being translated to produce the encoded Snu13 polypeptide in vitro, in vivo, in situ or ex vivo.
  • the polynucleotide of the invention (e.g., a RNA, e.g., an mRNA) comprises a sequence-optimized nucleotide sequence (e.g., an ORF) encoding a Snu13 polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), wherein the polynucleotide comprises at least one chemically modified nucleobase, e.g., N1-methylpseudouracil or 5-methoxyuracil.
  • all uracils in the polynucleotide are N1-methylpseudouracils.
  • all uracils in the polynucleotide are 5-methoxyuracils.
  • the polynucleotide further comprises a miRNA binding site, e.g., a miRNA binding site that binds to miR-142 and/or a miRNA binding site that binds to miR-126.
  • the polynucleotide e.g., a RNA, e.g., an mRNA
  • a delivery agent comprising, e.g., a compound having the Formula (I), e.g., Compound II or Compound B; a compound having the Formula (III), (IV), (V), or (VI), e.g., Compound VI or Compound I, or any combination thereof.
  • the delivery agent comprises an ionizable amino lipid (e.g., Compound II, VI, or B), a helper lipid (e.g., DSPC), a sterol (e.g., Cholesterol), and a PEG lipid (e.g., Compound I or PEG-DMG), e.g., with a mole ratio in the range of about (i) 40-50 mol% ionizable amino lipid (e.g., Compound II, VI, or B), optionally 45-50 mol% ionizable amino lipid, for example, 45-46 mol%, 46-47 mol%, 47-48 mol%, 48-49 mol%, or 49-50 mol% for example about 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol%, 48.5 mol%, 49 mol%, or 49.5 mol%; (ii) 30-45
  • the delivery agent comprises Compound II, Cholesterol, DSPC, and Compound I.
  • the polynucleotide of the disclosure is an mRNA that comprises a 5′-terminal cap (e.g., Cap1, e.g., m 7 Gp-ppGm-A), a 5′UTR comprising the nucleotide sequence of SEQ ID NO:50, a nucleotide sequence (e.g., an ORF, e.g., any one of SEQ ID NOs: 300-317) encoding a Snu13 polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), a 3′UTR (e.g., SEQ ID NO:108), and a poly A tail (e.g., about 100 nt in length, e.g., SEQ ID NO:195), wherein all uracils in the polynucleotide are N1-methylpse
  • a 5′-terminal cap
  • the delivery agent is an LNP.
  • the delivery agent comprises Compound II or Compound VI as the ionizable amino lipid and PEG-DMG or Compound I as the PEG lipid.
  • the polynucleotide of the disclosure is an mRNA that comprises a 5′-terminal cap (e.g., Cap1, e.g., m 7 Gp-ppGm-A), a 5′UTR (e.g., SEQ ID NO:50), the ORF sequence of any one of SEQ ID NOs:300-317, a 3′UTR (e.g., SEQ ID NO:108), and a poly A tail (e.g., about 100 nt in length, e.g., SEQ ID NO:195), wherein all uracils in the polynucleotide are N1-methylpseudouracils or 5- methoxyuracil.
  • a 5′-terminal cap e.g., Cap1, e.g.,
  • the delivery agent is an LNP.
  • the delivery agent comprises Compound II or Compound VI as the ionizable amino lipid and PEG-DMG or Compound I as the PEG lipid.
  • the polynucleotide of the disclosure is an mRNA that comprises a 5′-terminal cap (e.g., Cap1, e.g., m 7 Gp-ppGm-A), a 5′UTR (e.g., SEQ ID NO:50), a nucleotide sequence (e.g., an ORF, e.g., any one of SEQ ID NOs:300-317) encoding a Snu13 polypeptide, a 3′UTR comprising the nucleotide sequence of SEQ ID NO:108, and a poly A tail (e.g., about 100 nt in length, e.g., SEQ ID NO:195), wherein all uracils in the polynucle
  • a 5′-terminal cap e.g
  • the delivery agent is an LNP.
  • the delivery agent comprises Compound II or Compound VI as the ionizable amino lipid and PEG-DMG or Compound I as the PEG lipid.
  • the polynucleotides (e.g., a RNA, e.g., an mRNA) of the invention can also comprise nucleotide sequences that encode additional features that facilitate trafficking of the encoded polypeptides to therapeutically relevant sites.
  • One such feature that aids in protein trafficking is the signal sequence, or targeting sequence.
  • the polynucleotide e.g., a RNA, e.g., an mRNA
  • a nucleotide sequence e.g., an ORF
  • the "signal sequence” or “signal peptide” is a polynucleotide or polypeptide, respectively, which is from about 30-210, e.g., about 45-80 or 15-60 nucleotides (e.g., about 20, 30, 40, 50, 60, or 70 amino acids) in length that, optionally, is incorporated at the 5′ (or N-terminus) of the coding region or the polypeptide, respectively. Addition of these sequences results in trafficking the encoded polypeptide to a desired site, such as the endoplasmic reticulum or the mitochondria through one or more targeting pathways.
  • a desired site such as the endoplasmic reticulum or the mitochondria through one or more targeting pathways.
  • the polynucleotide of the invention comprises a nucleotide sequence encoding a Snu13 polypeptide, wherein the nucleotide sequence further comprises a 5′ nucleic acid sequence encoding a heterologous signal peptide. 4. Fusion Proteins [00122]
  • the polynucleotide of the invention e.g., a RNA, e.g., an mRNA
  • polynucleotides of the invention comprise a single ORF encoding a Snu13 polypeptide, a functional fragment, or a variant thereof.
  • two or more polypeptides of interest can be genetically fused, i.e., two or more polypeptides can be encoded by the same ORF.
  • the polynucleotide can comprise a nucleic acid Attorney Docket No.45817-0124WO1 / MTX959.20 sequence encoding a linker (e.g., a G4S (SEQ ID NO: 200) peptide linker or another linker known in the art) between two or more polypeptides of interest.
  • a polynucleotide of the invention e.g., a RNA, e.g., an mRNA
  • a polynucleotide of the invention can comprise two, three, four, or more ORFs, each expressing a polypeptide of interest.
  • Linkers and Cleavable Peptides [00124]
  • the mRNAs of the disclosure encode more than one Snu13 domain or a heterologous domain, referred to herein as multimer constructs.
  • the mRNA further encodes a linker located between each domain.
  • the linker can be, for example, a cleavable linker or protease-sensitive linker.
  • the linker is selected from the group consisting of F2A linker, P2A linker, T2A linker, E2A linker, and combinations thereof.
  • This family of self-cleaving peptide linkers referred to as 2A peptides, has been described in the art (see for example, Kim, J.H. et al. (2011) PLoS ONE 6:e18556).
  • the linker is an F2A linker.
  • the linker is a GGGS (SEQ ID NO: 201) linker.
  • the multimer construct contains three domains with intervening linkers, having the structure: domain-linker-domain-linker-domain e.g., Snu13 domain-linker- Snu13 domain-linker- Snu13 domain.
  • the cleavable linker is an F2A linker (e.g., having the amino acid sequence GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:189)).
  • the cleavable linker is a T2A linker (e.g., having the amino acid sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO:190)), a P2A linker (e.g., having the amino acid sequence GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:191)) or an E2A linker (e.g., having the amino acid sequence GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO:186)).
  • T2A linker e.g., having the amino acid sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO:190)
  • P2A linker e.g., having the amino acid sequence GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:191)
  • an E2A linker e.g., having the amino acid sequence GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO:186)
  • the construct design yields Attorney Docket No.45817-0124WO1 / MTX959.20 approximately equimolar amounts of intrabody and/or domain thereof encoded by the constructs of the invention.
  • the self-cleaving peptide may be, but is not limited to, a 2A peptide.
  • 2A peptides are known and available in the art and may be used, including e.g., the foot and mouth disease virus (FMDV) 2A peptide, the equine rhinitis A virus 2A peptide, the Thosea asigna virus 2A peptide, and the porcine teschovirus-12A peptide.
  • FMDV foot and mouth disease virus
  • 2A peptides are used by several viruses to generate two proteins from one transcript by ribosome-skipping, such that a normal peptide bond is impaired at the 2A peptide sequence, resulting in two discontinuous proteins being produced from one translation event.
  • the 2A peptide may have the protein sequence of SEQ ID NO: 191, fragments or variants thereof.
  • the 2A peptide cleaves between the last glycine and last proline.
  • the polynucleotides of the present invention may include a polynucleotide sequence encoding the 2A peptide having the protein sequence of fragments or variants of SEQ ID NO: 191.
  • a polynucleotide sequence encoding the 2A peptide is:GGAAGCGGAGCUACUAACUUCAGCCUGCUGAAGCAGGCUGGAGACGU GGAGGAGAACCCUGGACCU (SEQ ID NO:187).
  • a 2A peptide is encoded by the following sequence: 5′- UCCGGACUCAGAUCCGGGGAUCUCAAAAUUGUCGCUCCUGUCAAACAA ACUCUUAACUUUGAUUUACUCAAACUGGCUGGGGAUGUAGAAAGCAAU CCAGGUCCACUC-3′(SEQ ID NO: 188).
  • the polynucleotide sequence of the 2A peptide may be modified or codon optimized by the methods described herein and/or are known in the art. [00127] In one embodiment, this sequence may be used to separate the coding regions of two or more polypeptides of interest.
  • the sequence encoding the F2A peptide may be between a first coding region A and a second coding region B (A-F2Apep-B).
  • A-F2Apep-B The presence of the F2A peptide results in the cleavage of the one long protein between the glycine and the proline at the end of the F2A peptide sequence (NPGP (SEQ ID NO:205) is cleaved to result in NPG and P) thus creating separate protein A (with 21 amino acids of the F2A peptide attached, ending with NPG) and separate protein B (with 1 amino acid, P, of the F2A peptide Attorney Docket No.45817-0124WO1 / MTX959.20 attached).
  • Protein A and protein B may be the same or different peptides or polypeptides of interest (e.g., a Snu13 polypeptide described herein). 5.
  • the polynucleotide e.g., a RNA, e.g., an mRNA
  • sequence optimized is sequence optimized.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding a Snu13 polypeptide, optionally, a nucleotide sequence (e.g., an ORF) encoding another polypeptide of interest, a 5′-UTR, a 3′-UTR, the 5′ UTR or 3′ UTR optionally comprising at least one microRNA binding site, optionally a nucleotide sequence encoding a linker, a polyA tail, or any combination thereof), in which the ORF(s) are sequence optimized.
  • a nucleotide sequence e.g., an ORF
  • a sequence-optimized nucleotide sequence e.g., a codon-optimized mRNA sequence encoding a Snu13 polypeptide, is a sequence comprising at least one synonymous nucleobase substitution with respect to a reference sequence (e.g., a wild type nucleotide sequence encoding a Snu13 polypeptide).
  • a reference sequence e.g., a wild type nucleotide sequence encoding a Snu13 polypeptide.
  • a reference sequence encoding polyserine uniformly encoded by UCU codons can be sequence-optimized by having 100% of its nucleobases substituted (for each codon, U in position 1 replaced by A, C in position 2 replaced by G, and U in position 3 replaced by C) to yield a sequence encoding polyserine which would be uniformly encoded by AGC codons.
  • the percentage of sequence identity obtained from a global pairwise alignment between the reference polyserine nucleic acid sequence and the sequence- optimized polyserine nucleic acid sequence would be 0%.
  • the protein products from both sequences would be 100% identical.
  • results can include, e.g., matching codon frequencies in certain tissue targets and/or host organisms to ensure proper folding; biasing G/C content to increase mRNA stability or reduce secondary structures; minimizing tandem repeat codons or base runs that can impair gene construction or expression; customizing transcriptional and translational control regions; inserting or removing protein trafficking sequences; removing/adding post translation modification sites in an encoded protein (e.g., glycosylation sites); adding, removing or shuffling protein domains; inserting or deleting restriction sites; modifying ribosome binding sites and mRNA degradation sites; adjusting translational rates to allow the various domains of the protein to fold properly; and/or reducing or eliminating problem secondary structures within the polynucleotide.
  • an encoded protein e.g., glycosylation sites
  • adding, removing or shuffling protein domains inserting or deleting restriction sites; modifying ribosome binding sites and mRNA degradation sites; adjusting translational rates to allow the various domains of the protein to fold
  • Sequence optimization tools, algorithms and services are known in the art, non- limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods. [00132] Codon options for each amino acid are given in Table 3. [00133] Table 3.
  • SECIS of the invention comprises a sequence-optimized nucleotide sequence (e.g., an ORF) encoding a Snu13 polypeptide, a functional fragment, or a variant thereof, wherein the Snu13 polypeptide, functional fragment, or a variant thereof encoded by the sequence-optimized nucleotide sequence has improved properties (e.g., compared to a Snu13 polypeptide, functional fragment, or a variant thereof encoded by a reference nucleotide sequence that is not sequence optimized), e.g., improved properties related to expression efficacy after administration in vivo.
  • sequence-optimized nucleotide sequence e.g., an ORF
  • the Snu13 polypeptide, functional fragment, or a variant thereof encoded by the sequence-optimized nucleotide sequence has improved properties (e.g., compared to a Snu13 polypeptide, functional fragment, or a variant thereof encoded by a reference nucleotide sequence
  • Such properties include, but are not limited to, improving nucleic acid stability (e.g., mRNA stability), increasing translation efficacy in the target tissue, reducing the number of truncated proteins expressed, improving the folding or prevent misfolding of the expressed proteins, reducing toxicity of the expressed products, reducing cell death caused by the expressed products, increasing and/or decreasing protein aggregation.
  • nucleic acid stability e.g., mRNA stability
  • increasing translation efficacy in the target tissue reducing the number of truncated proteins expressed, improving the folding or prevent misfolding of the expressed proteins, reducing toxicity of the expressed products, reducing cell death caused by the expressed products, increasing and/or decreasing protein aggregation.
  • sequence-optimized nucleotide sequence (e.g., an ORF) is codon optimized for expression in human subjects, having structural and/or chemical features that avoid one or more of the problems in the art, for example, features which are useful for optimizing formulation and delivery of nucleic acid-based therapeutics while retaining structural and functional integrity; overcoming a threshold of expression; improving expression rates; half-life and/or protein concentrations; optimizing protein localization; and avoiding deleterious bio- responses such as the immune response and/or degradation pathways.
  • an ORF codon optimized for expression in human subjects, having structural and/or chemical features that avoid one or more of the problems in the art, for example, features which are useful for optimizing formulation and delivery of nucleic acid-based therapeutics while retaining structural and functional integrity; overcoming a threshold of expression; improving expression rates; half-life and/or protein concentrations; optimizing protein localization; and avoiding deleterious bio- responses such as the immune response and/or degradation pathways.
  • the polynucleotides of the invention comprise a nucleotide sequence (e.g., a nucleotide sequence (e.g., an ORF) encoding a Snu13 polypeptide, a nucleotide sequence (e.g., an ORF) encoding another polypeptide of interest, a 5′-UTR, a 3′-UTR, a microRNA binding site, a nucleic acid sequence encoding a linker, or any combination thereof) that is sequence-optimized according to a method comprising: Attorney Docket No.45817-0124WO1 / MTX959.20 (i) substituting at least one codon in a reference nucleotide sequence (e.g., an ORF encoding a Snu13 polypeptide) with an alternative codon to increase or decrease uridine content to generate a uridine-modified sequence; (ii) substituting at least one codon in a reference
  • the sequence-optimized nucleotide sequence (e.g., an ORF encoding a Snu13 polypeptide) has at least one improved property with respect to the reference nucleotide sequence.
  • the sequence optimization method is multiparametric and comprises one, two, three, four, or more methods disclosed herein and/or other optimization methods known in the art.
  • Features, which can be considered beneficial in some embodiments of the invention can be encoded by or within regions of the polynucleotide and such regions can be upstream (5′) to, downstream (3′) to, or within the region that encodes the Snu13 polypeptide.
  • the polynucleotide of the invention comprises a 5′ UTR, a 3′ UTR and/or a microRNA binding site.
  • the polynucleotide comprises two or more 5′ UTRs and/or 3′ UTRs, which can be the same or different sequences. In some embodiments, the polynucleotide comprises two or more microRNA binding sites, which can be the same or different sequences. Any portion of the 5′ UTR, 3′ UTR, and/or microRNA binding site, including none, can be Attorney Docket No.45817-0124WO1 / MTX959.20 sequence-optimized and can independently contain one or more different structural or chemical modifications, before and/or after sequence optimization.
  • the polynucleotide is reconstituted and transformed into a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes.
  • a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes.
  • the optimized polynucleotide can be reconstituted and transformed into chemically competent E. coli, yeast, neurospora, maize, drosophila, etc. where high copy plasmid-like or chromosome structures occur by methods described herein. 6.
  • the polynucleotide of the invention comprises a sequence-optimized nucleotide sequence encoding a Snu13 polypeptide disclosed herein.
  • the polynucleotide of the invention comprises an open reading frame (ORF) encoding a Snu13 polypeptide, wherein the ORF has been sequence optimized.
  • ORF open reading frame
  • Exemplary sequence-optimized nucleotide sequences encoding a Snu13 polypeptide are set forth in SEQ ID NOs:300-317.
  • a polynucleotide of the present disclosure for example a polynucleotide comprising an mRNA nucleotide sequence encoding a Snu13 polypeptide, comprises from 5′ to 3′ end: (i) a 5′ cap such as provided herein, for example, m 7 Gp-ppGm-A; (ii) a 5′ UTR comprising a nucleotide sequence set forth in Table 4 (e.g., SEQ ID NO:50); (iii) an open reading frame encoding a polypeptide comprising a Snu13 polypeptide, e.g., a sequence optimized nucleic acid sequence encoding Snu13 set forth as any one of SEQ ID NOs:300-317; (iv) at least one stop codon (if not present at 5′ termin
  • the polynucleotide of the invention comprises a sequence-optimized nucleotide sequence encoding a Snu13 polypeptide disclosed herein. In some embodiments, the polynucleotide of the invention comprises an ORF encoding a Snu13 polypeptide, wherein the ORF has been sequence optimized.
  • sequence-optimized nucleotide sequences disclosed herein are distinct from the corresponding wild type nucleotide acid sequences and from other known sequence-optimized nucleotide sequences, e.g., these sequence-optimized nucleic acids have unique compositional characteristics.
  • the percentage of uracil or thymine nucleobases in a sequence-optimized nucleotide sequence e.g., encoding a Snu13 polypeptide, a functional fragment, or a variant thereof
  • Such a sequence is referred to as a uracil-modified or thymine-modified sequence.
  • the percentage of uracil or thymine content in a nucleotide sequence can be determined by dividing the number of uracils or thymines in a sequence by the total number of nucleotides and multiplying by 100.
  • the sequence- optimized nucleotide sequence has a lower uracil or thymine content than the uracil or thymine content in the reference wild-type sequence.
  • the uracil or thymine content in a sequence-optimized nucleotide sequence of the invention is greater than the uracil or thymine content in the reference wild-type sequence and still maintain beneficial effects, e.g., increased expression and/or reduced Toll-Like Receptor (TLR) response when compared to the reference wild- type sequence.
  • TLR Toll-Like Receptor
  • Codon optimization may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize Attorney Docket No.45817-0124WO1 / MTX959.20 transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide.
  • Codon optimization tools, algorithms and services are known in the art - non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods.
  • the open reading frame (ORF) sequence is optimized using optimization algorithms. 7.
  • the polynucleotide e.g., a RNA, e.g., an mRNA
  • a sequence optimized nucleic acid disclosed herein encoding a Snu13 polypeptide can be tested to determine whether at least one nucleic acid sequence property (e.g., stability when exposed to nucleases) or expression property has been improved with respect to the non-sequence optimized nucleic acid.
  • expression property refers to a property of a nucleic acid sequence either in vivo (e.g., translation efficacy of a synthetic mRNA after administration to a subject in need thereof) or in vitro (e.g., translation efficacy of a synthetic mRNA tested in an in vitro model system).
  • Expression properties include but are not limited to the amount of protein produced by an mRNA encoding a Snu13 polypeptide after administration, and the amount of soluble or otherwise functional protein produced.
  • sequence optimized nucleic acids disclosed herein can be evaluated according to the viability of the cells expressing a protein encoded by a sequence optimized nucleic acid sequence (e.g., a RNA, e.g., an mRNA) encoding a Snu13 polypeptide disclosed herein.
  • a sequence optimized nucleic acid sequence e.g., a RNA, e.g., an mRNA
  • a plurality of sequence optimized nucleic acids disclosed herein e.g., a RNA, e.g., an mRNA
  • a RNA e.g., an mRNA
  • the desired property of the polynucleotide is an intrinsic property of the nucleic acid sequence.
  • the nucleotide sequence (e.g., a RNA, e.g., an mRNA) can be sequence optimized for in vivo or in vitro stability.
  • the nucleotide sequence can be sequence optimized for expression in a given target tissue or cell.
  • the nucleic acid sequence is sequence optimized to increase its plasma half-life by preventing its degradation by endo and exonucleases.
  • the nucleic acid sequence is sequence optimized to increase its resistance to hydrolysis in solution, for example, to lengthen the time that the sequence optimized nucleic acid or a pharmaceutical composition comprising the sequence optimized nucleic acid can be stored under aqueous conditions with minimal degradation.
  • the sequence optimized nucleic acid can be optimized to increase its resistance to hydrolysis in dry storage conditions, for example, to lengthen the time that the sequence optimized nucleic acid can be stored after lyophilization with minimal degradation.
  • the desired property of the polynucleotide is the level of expression of a Snu13 polypeptide encoded by a sequence optimized sequence disclosed herein. Protein expression levels can be measured using one or more expression systems. In some embodiments, expression can be measured in cell culture systems, e.g., CHO cells or HEK293 cells.
  • expression can be measured using in vitro expression systems prepared from extracts of living cells, e.g., rabbit reticulocyte lysates, or in vitro expression systems prepared by assembly of purified individual components.
  • in vitro expression systems prepared from extracts of living cells, e.g., rabbit reticulocyte lysates, or in vitro expression systems prepared by assembly of purified individual components.
  • the protein expression is measured in an in vivo system, e.g., mouse, rabbit, monkey, etc.
  • protein expression in solution form can be desirable.
  • a reference sequence can be sequence optimized to yield a sequence optimized nucleic acid sequence having optimized levels of expressed proteins in soluble form.
  • Levels of protein expression and other properties such as solubility, levels of aggregation, and the presence of truncation products can be measured according to methods known in the art, for example, using electrophoresis (e.g., native or SDS-PAGE) or chromatographic methods (e.g., HPLC, size exclusion chromatography, etc.).
  • electrophoresis e.g., native or SDS-PAGE
  • chromatographic methods e.g., HPLC, size exclusion chromatography, etc.
  • the expression of heterologous therapeutic proteins encoded by a nucleic acid sequence can have deleterious effects in the target tissue or cell, reducing protein yield, or reducing the quality of the expressed product (e.g., due to the presence of protein fragments or precipitation of the expressed protein in inclusion bodies), or causing toxicity.
  • the sequence optimization of a nucleic acid sequence disclosed herein e.g., a nucleic acid sequence encoding a Snu13 polypeptide, can be used to increase the viability of target cells expressing the protein encoded by the sequence optimized nucleic acid.
  • Heterologous protein expression can also be deleterious to cells transfected with a nucleic acid sequence for autologous or heterologous transplantation. Accordingly, in some embodiments of the present disclosure the sequence optimization of a nucleic acid sequence disclosed herein can be used to increase the viability of target cells expressing the protein encoded by the sequence optimized nucleic acid sequence. Changes in cell or tissue viability, toxicity, and other physiological reaction can be measured according to methods known in the art. Attorney Docket No.45817-0124WO1 / MTX959.20 d.
  • the administration of a sequence optimized nucleic acid encoding a Snu13 polypeptide, or a functional fragment thereof can trigger an immune response, which could be caused by (i) the therapeutic agent (e.g., an mRNA encoding a Snu13 polypeptide), or (ii) the expression product of such therapeutic agent (e.g., the Snu13 polypeptide encoded by the mRNA), or (iv) a combination thereof.
  • the therapeutic agent e.g., an mRNA encoding a Snu13 polypeptide
  • the expression product of such therapeutic agent e.g., the Snu13 polypeptide encoded by the mRNA
  • nucleic acid sequence e.g., an mRNA
  • sequence optimization of nucleic acid sequence can be used to decrease an immune or inflammatory response triggered by the administration of a nucleic acid encoding a Snu13 polypeptide or by the expression product of Snu13 encoded by such nucleic acid.
  • an inflammatory response can be measured by detecting increased levels of one or more inflammatory cytokines using methods known in the art, e.g., ELISA.
  • inflammatory cytokine refers to cytokines that are elevated in an inflammatory response.
  • inflammatory cytokines examples include interleukin-6 (IL-6), CXCL1 (chemokine (C-X-C motif) ligand 1; also known as GRO ⁇ , interferon- ⁇ (IFN ⁇ ), tumor necrosis factor ⁇ (TNF ⁇ ), interferon ⁇ -induced protein 10 (IP-10), or granulocyte-colony stimulating factor (G-CSF).
  • IL-6 interleukin-6
  • CXCL1 chemokine (C-X-C motif) ligand 1
  • GRO ⁇ interferon- ⁇
  • IFN ⁇ interferon- ⁇
  • TNF ⁇ tumor necrosis factor ⁇
  • IP-10 interferon ⁇ -induced protein 10
  • G-CSF granulocyte-colony stimulating factor
  • the term inflammatory cytokines includes also other cytokines associated with inflammatory responses known in the art, e.g., interleukin-1 (IL-1), interleukin-8 (IL-8), interleukin
  • the polynucleotide e.g., a RNA, e.g., an mRNA
  • the polynucleotide of the invention comprises a chemically modified nucleobase, for example, a chemically modified uracil, e.g., pseudouracil, N1-methylpseudouracil, 5-methoxyuracil, or the like.
  • the mRNA is a uracil-modified sequence comprising an ORF encoding a Snu13 polypeptide, wherein the mRNA comprises a chemically modified nucleobase, for example, a chemically modified uracil, e.g., pseudouracil, N1-methylpseudouracil, or 5-methoxyuracil.
  • a chemically modified uracil e.g., pseudouracil, N1-methylpseudouracil, or 5-methoxyuracil.
  • Attorney Docket No.45817-0124WO1 / MTX959.20 [00163]
  • the modified uracil base is connected to a ribose sugar, as it is in polynucleotides, the resulting modified nucleoside or nucleotide is referred to as modified uridine.
  • uracil in the polynucleotide is at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least 90%, at least 95%, at least 99%, or about 100% modified uracil. In one embodiment, uracil in the polynucleotide is at least 95% modified uracil. In another embodiment, uracil in the polynucleotide is 100% modified uracil. [00164] In embodiments where uracil in the polynucleotide is at least 95% modified uracil overall uracil content can be adjusted such that an mRNA provides suitable protein expression levels while inducing little to no immune response.
  • the uracil content of the ORF is between about 100% and about 150%, between about 100% and about 110%, between about 105% and about 115%, between about 110% and about 120%, between about 115% and about 125%, between about 120% and about 130%, between about 125% and about 135%, between about 130% and about 140%, between about 135% and about 145%, between about 140% and about 150% of the theoretical minimum uracil content in the corresponding wild- type ORF (%UTM).
  • the uracil content of the ORF is between about 121% and about 136% or between 123% and 134% of the %U TM .
  • the uracil content of the ORF encoding a Snu13 polypeptide is about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, or about 150% of the %UTM.
  • uracil can refer to modified uracil and/or naturally occurring uracil.
  • the uracil content in the ORF of the mRNA encoding a Snu13 polypeptide of the invention is less than about 30%, about 25%, about 20%, about 15%, or about 10% of the total nucleobase content in the ORF.
  • the uracil content in the ORF is between about 10% and about 20% of the total nucleobase content in the ORF. In other embodiments, the uracil content in the ORF is between about 10% and about 25% of the total nucleobase content in the ORF. In one embodiment, the uracil content in the ORF of the mRNA encoding a Snu13 polypeptide is less than about 20% of the total nucleobase content Attorney Docket No.45817-0124WO1 / MTX959.20 in the open reading frame. In this context, the term "uracil" can refer to modified uracil and/or naturally occurring uracil.
  • the ORF of the mRNA encoding a Snu13 polypeptide having modified uracil and adjusted uracil content has increased Cytosine (C), Guanine (G), or Guanine/Cytosine (G/C) content (absolute or relative).
  • the overall increase in C, G, or G/C content (absolute or relative) of the ORF is at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 10%, at least about 15%, at least about 20%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% relative to the G/C content (absolute or relative) of the wild-type ORF.
  • the G, the C, or the G/C content in the ORF is less than about 100%, less than about 90%, less than about 85%, or less than about 80% of the theoretical maximum G, C, or G/C content of the corresponding wild type nucleotide sequence encoding the Snu13 polypeptide (%G TMX ; %C TMX , or %G/C TMX ).
  • the increases in G and/or C content (absolute or relative) described herein can be conducted by replacing synonymous codons with low G, C, or G/C content with synonymous codons having higher G, C, or G/C content.
  • the increase in G and/or C content is conducted by replacing a codon ending with U with a synonymous codon ending with G or C.
  • the ORF of the mRNA encoding a Snu13 polypeptide of the invention comprises modified uracil and has an adjusted uracil content containing less uracil pairs (UU) and/or uracil triplets (UUU) and/or uracil quadruplets (UUUU) than the corresponding wild-type nucleotide sequence encoding the Snu13 polypeptide.
  • the ORF of the mRNA encoding a Snu13 polypeptide of the invention contains no uracil pairs and/or uracil triplets and/or uracil quadruplets. In some embodiments, uracil pairs and/or uracil triplets and/or uracil quadruplets are reduced below a certain threshold, e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 occurrences in the ORF of the mRNA encoding the Snu13 polypeptide.
  • a certain threshold e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 occurrences in the ORF of the mRNA encoding the Snu13 polypeptide.
  • the ORF of the mRNA encoding the Snu13 polypeptide of the invention contains less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 non- Attorney Docket No.45817-0124WO1 / MTX959.20 phenylalanine uracil pairs and/or triplets.
  • the ORF of the mRNA encoding the Snu13 polypeptide contains no non-phenylalanine uracil pairs and/or triplets.
  • the ORF of the mRNA encoding a Snu13 polypeptide of the invention comprises modified uracil and has an adjusted uracil content containing less uracil-rich clusters than the corresponding wild-type nucleotide sequence encoding the Snu13 polypeptide.
  • the ORF of the mRNA encoding the Snu13 polypeptide of the invention contains uracil-rich clusters that are shorter in length than corresponding uracil-rich clusters in the corresponding wild-type nucleotide sequence encoding the Snu13 polypeptide.
  • alternative lower frequency codons are employed.
  • the ORF also has adjusted uracil content, as described above.
  • at least one codon in the ORF of the mRNA encoding the Snu13 polypeptide is substituted with an alternative codon having a codon frequency lower than the codon frequency of the substituted codon in the synonymous codon set.
  • the Snu13 polypeptide-encoding ORF comprising an adjusted uracil content exhibits expression levels of Snu13 when administered to a mammalian cell that are higher than expression levels of Snu13 from the corresponding wild-type mRNA.
  • the mammalian cell is a mouse cell, a rat cell, or a rabbit cell.
  • the mammalian cell is a monkey cell or a human cell.
  • the human cell is a HeLa cell, a BJ fibroblast cell, or a peripheral blood mononuclear cell (PBMC).
  • PBMC peripheral blood mononuclear cell
  • Snu13 is expressed at a level higher than expression levels of Snu13 from the corresponding wild-type mRNA when the mRNA encoding Snu13 is Attorney Docket No.45817-0124WO1 / MTX959.20 administered to a mammalian cell in vivo.
  • the mRNA is administered to mice, rabbits, rats, monkeys, or humans. In one embodiment, mice are null mice.
  • the mRNA is administered to mice in an amount of about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, or 0.2 mg/kg or about 0.5 mg/kg. In some embodiments, the mRNA is administered intravenously or intramuscularly. In other embodiments, the Snu13 polypeptide is expressed when the mRNA is administered to a mammalian cell in vitro. In some embodiments, the expression is increased by at least about 2-fold, at least about 5-fold, at least about 10- fold, at least about 50-fold, at least about 500-fold, at least about 1500-fold, or at least about 3000-fold.
  • the expression is increased by at least about 10%, about 20%, about 30%, about 40%, about 50%, 60%, about 70%, about 80%, about 90%, or about 100%.
  • the Snu13 polypeptide-encoding ORF comprising an adjusted uracil content exhibits increased stability.
  • the mRNA exhibits increased stability in a cell relative to the stability of a corresponding wild-type mRNA under the same conditions.
  • the mRNA exhibits increased stability including resistance to nucleases, thermal stability, and/or increased stabilization of secondary structure.
  • increased stability exhibited by the mRNA is measured by determining the half-life of the mRNA (e.g., in a plasma, serum, cell, or tissue sample) and/or determining the area under the curve (AUC) of the protein expression by the mRNA over time (e.g., in vitro or in vivo).
  • An mRNA is identified as having increased stability if the half-life and/or the AUC is greater than the half-life and/or the AUC of a corresponding wild- type mRNA under the same conditions.
  • the mRNA of the present invention induces a detectably lower immune response (e.g., innate or acquired) relative to the immune response induced by a corresponding wild-type mRNA under the same conditions.
  • the mRNA of the present disclosure induces a detectably lower immune response (e.g., innate or acquired) relative to the immune response induced by an mRNA that encodes for a Snu13 polypeptide but does not comprise modified uracil under the same conditions, or relative to the immune response induced by an mRNA that encodes for a Snu13 and that comprises modified uracil but that does not Attorney Docket No.45817-0124WO1 / MTX959.20 have adjusted uracil content under the same conditions.
  • the innate immune response can be manifested by increased expression of pro-inflammatory cytokines, activation of intracellular PRRs (RIG-I, MDA5, etc.), cell death, and/or termination or reduction in protein translation.
  • a reduction in the innate immune response can be measured by expression or activity level of Type 1 interferons (e.g., IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , and IFN- ⁇ ) or the expression of interferon-regulated genes such as the toll-like receptors (e.g., TLR7 and TLR8), and/or by decreased cell death following one or more administrations of the mRNA of the invention into a cell.
  • Type 1 interferons e.g., IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , and IFN- ⁇
  • interferon-regulated genes e.g., TLR7 and TLR8
  • the expression of Type-1 interferons by a mammalian cell in response to the mRNA of the present disclosure is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or greater than 99.9% relative to a corresponding wild-type mRNA, to an mRNA that encodes a Snu13 polypeptide but does not comprise modified uracil, or to an mRNA that encodes a Snu13 polypeptide, respectively, and that comprises modified uracil but that does not have adjusted uracil content.
  • the interferon is IFN- ⁇ .
  • cell death frequency caused by administration of mRNA of the present disclosure to a mammalian cell is 10%, 25%, 50%, 75%, 85%, 90%, 95%, or over 95% less than the cell death frequency observed with a corresponding wild-type mRNA, an mRNA that encodes for a Snu13 polypeptide but does not comprise modified uracil, or an mRNA that encodes for a Snu13 polypeptide, respectively, and that comprises modified uracil but that does not have adjusted uracil content.
  • the mammalian cell is a BJ fibroblast cell. In other embodiments, the mammalian cell is a splenocyte.
  • the mammalian cell is that of a mouse or a rat. In other embodiments, the mammalian cell is that of a human.
  • the mRNA of the present disclosure does not substantially induce an innate immune response of a mammalian cell into which the mRNA is introduced.
  • Attorney Docket No.45817-0124WO1 / MTX959.20 9. Methods for Modifying Polynucleotides [00174]
  • the disclosure includes modified polynucleotides comprising a polynucleotide described herein (e.g., a polynucleotide, e.g. mRNA, comprising a nucleotide sequence encoding a Snu13 polypeptide).
  • modified polynucleotides can be chemically modified and/or structurally modified.
  • modified polynucleotides can be referred to as "modified polynucleotides.”
  • the present disclosure provides for modified nucleosides and nucleotides of a polynucleotide (e.g., RNA polynucleotides, such as mRNA polynucleotides) encoding a Snu13 polypeptide.
  • nucleoside refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as "nucleobase").
  • organic base e.g., a purine or pyrimidine
  • nucleobase also referred to herein as “nucleobase”
  • nucleotide refers to a nucleoside including a phosphate group. Modified nucleotides can be synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Polynucleotides can comprise a region or regions of linked nucleosides.
  • modified polynucleotides disclosed herein can comprise various distinct modifications.
  • the modified polynucleotides contain one, two, or more (optionally different) nucleoside or nucleotide modifications.
  • a modified polynucleotide, introduced to a cell can exhibit one or more desirable properties, e.g., improved protein expression, reduced immunogenicity, or reduced degradation in the cell, as compared to an unmodified polynucleotide.
  • a polynucleotide of the present invention e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide
  • a "structural" modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide without significant chemical modification to the nucleotides Attorney Docket No.45817-0124WO1 / MTX959.20 themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications.
  • compositions of the present disclosure comprise, in some embodiments, at least one nucleic acid (e.g., RNA) having an open reading frame encoding Snu13, wherein the nucleic acid comprises nucleotides and/or nucleosides that can be standard (unmodified) or modified as is known in the art.
  • nucleic acid e.g., RNA
  • nucleotides and nucleosides of the present disclosure comprise modified nucleotides or nucleosides.
  • modified nucleotides and nucleosides can be naturally-occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides.
  • modifications can include those at the sugar, backbone, or nucleobase portion of the nucleotide and/or nucleoside as are recognized in the art.
  • a naturally-occurring modified nucleotide or nucleotide of the disclosure is one as is generally known or recognized in the art.
  • Non-limiting examples of such naturally occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database.
  • a non-naturally occurring modified nucleotide or nucleoside of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published US application Nos.
  • RNA e.g., mRNA
  • at least one RNA (e.g., mRNA) of the present disclosure is not chemically modified and comprises the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine.
  • nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues such as those present in transcribed RNA (e.g. A, G, C, or U).
  • nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides such as those present in DNA (e.g. dA, dG, dC, or dT).
  • nucleic acids of the disclosure can comprise standard nucleotides and nucleosides, naturally-occurring nucleotides and nucleosides, non-naturally-occurring nucleotides and nucleosides, or any combination thereof.
  • Nucleic acids of the disclosure e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids
  • Nucleic acids of the disclosure in some embodiments, comprise various (more than one) different types of standard and/or modified nucleotides and nucleosides.
  • a particular region of a nucleic acid contains one, two or more (optionally different) types of standard and/or modified nucleotides and nucleosides.
  • a modified RNA nucleic acid e.g., a modified mRNA nucleic acid
  • introduced to a cell or organism exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.
  • a modified RNA nucleic acid (e.g., a modified mRNA nucleic acid), introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response) relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.
  • Nucleic acids e.g., RNA nucleic acids, such as mRNA nucleic acids
  • Nucleic acids in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the nucleic acids to achieve desired functions or properties. The modifications may be present on internucleotide linkages, purine or pyrimidine bases, or sugars.
  • the modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a nucleic acid may be chemically modified.
  • the present disclosure provides for modified nucleosides and nucleotides of a nucleic acid (e.g., RNA nucleic acids, such as mRNA nucleic acids).
  • nucleoside refers to a compound containing a sugar molecule (e.g., a pentose or Attorney Docket No.45817-0124WO1 / MTX959.20 ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”).
  • an organic base e.g., a purine or pyrimidine
  • nucleobase also referred to herein as “nucleobase”.
  • nucleotide refers to a nucleoside, including a phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non- natural nucleosides.
  • Nucleic acids can comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the nucleic acids would comprise regions of nucleotides.
  • Modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non- standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures, such as, for example, in those nucleic acids having at least one chemical modification.
  • modified nucleobases in nucleic acids comprise N1-methyl-pseudouridine (m1 ⁇ ), 1-ethyl-pseudouridine (e1 ⁇ ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and/or pseudouridine ( ⁇ ).
  • modified nucleobases in nucleic acids comprise 5- methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5- methyl cytidine, and/or 5-methoxy cytidine.
  • the polyribonucleotide includes a combination of at least two (e.g., 2, 3, 4 or more) of any of the aforementioned modified nucleobases, including but not limited to chemical modifications.
  • a RNA nucleic acid of the disclosure comprises N1-methyl-pseudouridine (m1 ⁇ ) substitutions at one or more or all uridine positions of the nucleic acid.
  • m1 ⁇ N1-methyl-pseudouridine
  • MTX959.20 N1-methyl-pseudouridine substitutions at one or more or all uridine positions of the nucleic acid.
  • m1 ⁇ N1-methyl-pseudouridine substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid.
  • a RNA nucleic acid of the disclosure comprises pseudouridine ( ⁇ ) substitutions at one or more or all uridine positions of the nucleic acid.
  • a RNA nucleic acid of the disclosure comprises pseudouridine ( ⁇ ) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid.
  • a RNA nucleic acid of the disclosure comprises uridine at one or more or all uridine positions of the nucleic acid.
  • nucleic acids e.g., RNA nucleic acids, such as mRNA nucleic acids
  • RNA nucleic acids are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification.
  • a nucleic acid can be uniformly modified with N1-methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with N1-methyl-pseudouridine.
  • a nucleic acid can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.
  • the nucleic acids of the present disclosure may be partially or fully modified along the entire length of the molecule.
  • one or more or all or a given type of nucleotide may be uniformly modified in a nucleic acid of the disclosure, or in a predetermined sequence region thereof (e.g., in the mRNA including or excluding the polyA tail).
  • nucleotides X in a nucleic acid of the present disclosure are modified nucleotides, wherein X may be any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.
  • the nucleic acid may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or Attorney Docket No.45817-0124WO1 / MTX959.20 more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 20% to 70%, from 20% to 80%
  • the nucleic acids may contain at a minimum 1% and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides.
  • the nucleic acids may contain a modified pyrimidine such as a modified uracil or cytosine.
  • At least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the nucleic acid is replaced with a modified uracil (e.g., a 5-substituted uracil).
  • the modified uracil can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).
  • cytosine in the nucleic acid is replaced with a modified cytosine (e.g., a 5-substituted cytosine).
  • the modified cytosine can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).
  • Untranslated Regions are nucleic acid sections of a polynucleotide before a start codon (5′ UTR) and after a stop codon (3′ UTR) that are not translated.
  • a polynucleotide e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)
  • RNA e.g., a messenger RNA (mRNA)
  • RNA messenger RNA
  • ORF open reading frame
  • Snu13 polypeptide further comprises UTR (e.g., a 5′ UTR or functional fragment thereof, a 3′ UTR or functional fragment thereof, or a combination thereof).
  • a UTR (e.g., 5′ UTR or 3′ UTR) can be homologous or heterologous to the coding region in a polynucleotide.
  • the UTR is homologous to the ORF encoding the Snu13 polypeptide.
  • the UTR is heterologous to the ORF encoding the Snu13 polypeptide.
  • the polynucleotide comprises two or more 5′ UTRs or functional fragments thereof, each of which has the same or different nucleotide sequences.
  • the polynucleotide comprises two or more 3′ UTRs or functional fragments thereof, each of which has the same or different nucleotide sequences.
  • the 5′ UTR or functional fragment thereof, 3′ UTR or functional fragment thereof, or any combination thereof is sequence optimized.
  • the 5′UTR or functional fragment thereof, 3′ UTR or functional fragment thereof, or any combination thereof comprises at least one chemically modified nucleobase, e.g., N1-methylpseudouracil or 5-methoxyuracil.
  • UTRs can have features that provide a regulatory role, e.g., increased or decreased stability, localization and/or translation efficiency.
  • a polynucleotide comprising a UTR can be administered to a cell, tissue, or organism, and one or more regulatory features can be measured using routine methods.
  • a functional fragment of a 5′ UTR or 3′ UTR comprises one or more regulatory features of a full length 5′ or 3′ UTR, respectively.
  • Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or Attorney Docket No.45817-0124WO1 / MTX959.20 guanine) three bases upstream of the start codon (AUG), which is followed by another ‘G’.5′ UTRs also have been known to form secondary structures that are involved in elongation factor binding. [00206] By engineering the features typically found in abundantly expressed genes of specific target organs, one can enhance the stability and protein production of a polynucleotide.
  • liver-expressed mRNA such as albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII, can enhance expression of polynucleotides in hepatic cell lines or liver.
  • 5′UTR from other tissue-specific mRNA to improve expression in that tissue is possible for muscle (e.g., MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (e.g., Tie-1, CD36), for myeloid cells (e.g., C/EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), for leukocytes (e.g., CD45, CD18), for adipose tissue (e.g., CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (e.g., SP-A/B/C/D).
  • muscle e.g., MyoD, Myosin, Myoglobin, Myogenin, Herculin
  • endothelial cells e.g., Tie-1, CD36
  • myeloid cells e.g., C/E
  • UTRs are selected from a family of transcripts whose proteins share a common function, structure, feature or property.
  • an encoded polypeptide can belong to a family of proteins (i.e., that share at least one function, structure, feature, localization, origin, or expression pattern), which are expressed in a particular cell, tissue or at some time during development.
  • the UTRs from any of the genes or mRNA can be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide.
  • the 5′ UTR and the 3′ UTR can be heterologous.
  • the 5′ UTR can be derived from a different species than the 3′ UTR.
  • the 3′ UTR can be derived from a different species than the 5′ UTR.
  • Co-owned International Patent Application No. PCT/US2014/021522 (Publ. No. WO/2014/164253, incorporated herein by reference in its entirety) provides a listing of exemplary UTRs that can be utilized in the polynucleotide of the present invention as flanking regions to an ORF.
  • Additional exemplary UTRs of the application include, but are not limited to, one or more 5′UTR and/or 3′UTR derived from the nucleic acid sequence of: a globin, such as an ⁇ - or ⁇ -globin (e.g., a Xenopus, mouse, rabbit, or human globin); a Attorney Docket No.45817-0124WO1 / MTX959.20 strong Kozak translational initiation signal; a CYBA (e.g., human cytochrome b-245 ⁇ polypeptide); an albumin (e.g., human albumin7); a HSD17B4 (hydroxysteroid (17- ⁇ ) dehydrogenase); a virus (e.g., a tobacco etch virus (TEV), a Venezuelan equine encephalitis virus (VEEV), a Dengue virus, a cytomegalovirus (CMV) (e.g., CMV immediate early 1 (IE1)), a
  • the 5′ UTR is selected from the group consisting of a ⁇ -globin 5′ UTR; a 5′UTR containing a strong Kozak translational initiation signal; a cytochrome b-245 ⁇ polypeptide (CYBA) 5′ UTR; a hydroxysteroid (17- ⁇ ) dehydrogenase (HSD17B4) 5′ UTR; a Tobacco etch virus (TEV) 5′ UTR; a Vietnamese etch virus (TEV) 5′ UTR; a decielen equine encephalitis virus (TEEV) 5′ UTR; a 5′ proximal open reading frame of rubella virus (RV) RNA encoding nonstructural proteins; a Dengue virus (DEN) 5′ UTR; a heat shock protein 70 (Hsp70) 5′ UTR; a eIF4G 5′ UTR; a GLUT1 5′ UTR; functional fragments thereof and any combination thereof.
  • CYBA cytochrome b-2
  • the 3′ UTR is selected from the group consisting of a ⁇ -globin 3′ UTR; a CYBA 3′ UTR; an albumin 3′ UTR; a growth hormone (GH) 3′ UTR; a VEEV 3′ UTR; a hepatitis B virus (HBV) 3′ UTR; ⁇ -globin 3′UTR; a DEN 3′ Attorney Docket No.45817-0124WO1 / MTX959.20 UTR; a PAV barley yellow dwarf virus (BYDV-PAV) 3′ UTR; an elongation factor 1 ⁇ 1 (EEF1A1) 3′ UTR; a manganese superoxide dismutase (MnSOD) 3′ UTR; a ⁇ subunit of mitochondrial H(+)-ATP synthase ( ⁇ -mRNA) 3′ UTR; a GLUT13′ UTR; a MEF2A 3′ UTR; a ⁇ -
  • Wild-type UTRs derived from any gene or mRNA can be incorporated into the polynucleotides of the invention.
  • a UTR can be altered relative to a wild type or native UTR to produce a variant UTR, e.g., by changing the orientation or location of the UTR relative to the ORF; or by inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides.
  • variants of 5′ or 3′ UTRs can be utilized, for example, mutants of wild type UTRs, or variants wherein one or more nucleotides are added to or removed from a terminus of the UTR.
  • one or more synthetic UTRs can be used in combination with one or more non-synthetic UTRs. See, e.g., Mandal and Rossi, Nat. Protoc.2013 8(3):568-82, the contents of which are incorporated herein by reference in their entirety.
  • UTRs or portions thereof can be placed in the same orientation as in the transcript from which they were selected or can be altered in orientation or location. Hence, a 5′ and/or 3′ UTR can be inverted, shortened, lengthened, or combined with one or more other 5′ UTRs or 3′ UTRs.
  • the polynucleotide comprises multiple UTRs, e.g., a double, a triple or a quadruple 5′ UTR or 3′ UTR.
  • a double UTR comprises two copies of the same UTR either in series or substantially in series.
  • a double beta-globin 3′UTR can be used (see US2010/0129877, the contents of which are incorporated herein by reference in its entirety).
  • the polynucleotides of the invention can comprise combinations of features.
  • the ORF can be flanked by a 5′UTR that comprises a strong Kozak translational initiation signal and/or a 3′UTR comprising an oligo(dT) sequence for templated addition of a poly-A tail.
  • a 5′UTR can comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or Attorney Docket No.45817-0124WO1 / MTX959.20 different UTRs (see, e.g., US2010/0293625, herein incorporated by reference in its entirety).
  • Other non-UTR sequences can be used as regions or subregions within the polynucleotides of the invention.
  • the polynucleotide of the invention comprises an internal ribosome entry site (IRES) instead of or in addition to a UTR (see, e.g., Yakubov et al., Biochem. Biophys. Res. Commun.2010394(1):189-193, the contents of which are incorporated herein by reference in their entirety).
  • IRES internal ribosome entry site
  • the polynucleotide comprises an IRES instead of a 5′ UTR sequence.
  • the polynucleotide comprises an ORF and a viral capsid sequence.
  • the polynucleotide comprises a synthetic 5′ UTR in combination with a non-synthetic 3′ UTR.
  • the UTR can also include at least one translation enhancer polynucleotide, translation enhancer element, or translational enhancer elements (collectively, "TEE," which refers to nucleic acid sequences that increase the amount of polypeptide or protein produced from a polynucleotide.
  • TEE translation enhancer polynucleotide, translation enhancer element, or translational enhancer elements
  • the TEE can be located between the transcription promoter and the start codon.
  • the 5′ UTR comprises a TEE.
  • a TEE is a conserved element in a UTR that can promote translational activity of a nucleic acid such as, but not limited to, cap-dependent or cap-independent translation.
  • a.5′ UTR sequences [00221] 5′ UTR sequences are important for ribosome recruitment to the mRNA and have been reported to play a role in translation (Hinnebusch A, et al., (2016) Science, 352:6292: 1413-6).
  • a polynucleotide e.g., mRNA
  • mRNA comprising an open reading frame encoding a Snu13 polypeptide
  • polynucleotide has a 5′ Attorney Docket No.45817-0124WO1 / MTX959.20 UTR that confers an increased half-life, increased expression and/or increased activity of the polypeptide encoded by said polynucleotide, or of the polynucleotide itself.
  • a polynucleotide disclosed herein comprises: (a) a 5′-UTR (e.g., as provided in Table 4 or a variant or fragment thereof); (b) a coding region comprising a stop element (e.g., as described herein); and (c) a 3′-UTR (e.g., as described herein), and LNP compositions comprising the same.
  • the polynucleotide comprises a 5′-UTR comprising a sequence provided in Table 4 or a variant or fragment thereof (e.g., a functional variant or fragment thereof).
  • the polynucleotide comprises a 5′-UTR comprising the sequence of SEQ ID NO:50.
  • the polynucleotide having a 5′ UTR sequence provided in Table 4 or a variant or fragment thereof has an increase in the half-life of the polynucleotide, e.g., about 1.5-20-fold increase in half-life of the polynucleotide.
  • the increase in half-life is about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20-fold, or more.
  • the increase in half life is about 1.5-fold or more.
  • the increase in half life is about 2- fold or more.
  • the increase in half life is about 3-fold or more.
  • the increase in half life is about 4-fold or more.
  • the increase in half life is about 5-fold or more.
  • the polynucleotide having a 5′ UTR sequence provided in Table 4 or a variant or fragment thereof results in an increased level and/or activity, e.g., output, of the polypeptide encoded by the polynucleotide.
  • the 5′UTR results in about 1.5-20-fold increase in level and/or activity, e.g., output, of the polypeptide encoded by the polynucleotide.
  • the increase in level and/or activity is about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20-fold, or more.
  • the increase in level and/or activity is about 1.5-fold or more. In an embodiment, the increase in level and/or activity is about 2-fold or more. In an embodiment, the increase in level and/or activity is about 3-fold or more. In an embodiment, the increase in level and/or activity is about 4-fold or more. In an embodiment, the increase in level and/or activity is about 5-fold or more.
  • Attorney Docket No.45817-0124WO1 / MTX959.20 [00225] In an embodiment, the increase is compared to an otherwise similar polynucleotide which does not have a 5′ UTR, has a different 5′ UTR, or does not have a 5′ UTR described in Table 4 or a variant or fragment thereof.
  • the increase in half-life of the polynucleotide is measured according to an assay that measures the half-life of a polynucleotide.
  • the increase in level and/or activity, e.g., output, of the polypeptide encoded by the polynucleotide is measured according to an assay that measures the level and/or activity of a polypeptide.
  • the 5′ UTR comprises a sequence provided in Table 4 or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a 5′ UTR sequence provided in Table 4, or a variant or a fragment thereof.
  • the 5′ UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57 or SEQ ID NO: 58.
  • the 5′ UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 50.
  • the 5′ UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 51. In an embodiment, the 5′ UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 52. In an embodiment, the 5′ UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 53.
  • the 5′ UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 54. In an embodiment, the 5′ UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 55. In an embodiment, the 5′ UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 56.
  • the 5′ UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 57. In an embodiment, the 5′ UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 58. Attorney Docket No.45817-0124WO1 / MTX959.20 [00230] In an embodiment, the 5′ UTR comprises the sequence of SEQ ID NO:50. In an embodiment, the 5′ UTR consists of the sequence of SEQ ID NO:50.
  • a 5′ UTR sequence provided in Table 4 additionally has a first nucleotide which is an A. In an embodiment, a 5′ UTR sequence provided in Table 4 additionally has a first nucleotide which is a G.
  • Table 4 5′ UTR sequences SEQ ID Sequence Sequence NO: name A A U U A C A G C U A A A C Attorney Docket No.45817-0124WO1 / MTX959.20 A 3 GGAAAUCGCAAAAUUUUCUUUUCGCGUUAGAUUUCUUUUA GUUUUCUUUCAACUAGCAAGCUUUUGUUCUCGCCGCCGC n in G G G G U G C Attorney Docket No.45817-0124WO1 / MTX959.20 6 7 A18 GGAAACCCGCCCAAGCGACCCCAACAUAUCAGCAGUUGCC CAAUCCCAACUCCCAACACAAUCCCCAAGCAACGCCGCC G U A A G A A A A A A [ ] n an emo ment, te comprses a varant o Q : .
  • (N2)x is a uracil and x is 0. In an embodiment (N2)x is a uracil and x is 1. In an embodiment (N 2 ) x is a uracil and x is 2. In an embodiment (N2)x is a uracil and x is 3. In an embodiment, (N2)x is a uracil and x is 4. In an embodiment (N 2 ) x is a uracil and x is 5. [00235] In an embodiment, (N3)x is a guanine and x is 0. In an embodiment, (N3)x is a guanine and x is 1.
  • (N4)x is a cytosine and x is 0. In an embodiment, (N4)x is a cytosine and x is 1. [00237] In an embodiment (N5)x is a uracil and x is 0. In an embodiment (N5)x is a uracil and x is 1. In an embodiment (N 5 ) x is a uracil and x is 2. In an embodiment (N5)x is a uracil and x is 3. In an embodiment, (N5)x is a uracil and x is 4. In an embodiment (N 5 ) x is a uracil and x is 5. [00238] In an embodiment, N6 is a uracil.
  • N6 is a cytosine.
  • N 7 is a uracil.
  • N 7 is a guanine.
  • N8 is an adenine and x is 0.
  • N8 is an adenine and x is 1.
  • N 8 is a guanine and x is 0.
  • N 8 is a guanine and x is 1.
  • the 5′ UTR comprises a variant of SEQ ID NO:50.
  • the variant of SEQ ID NO: 50 comprises a sequence with at least Attorney Docket No.45817-0124WO1 / MTX959.20 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 50.
  • the variant of SEQ ID NO: 50 comprises a sequence with at least 50% identity to SEQ ID NO: 50.
  • the variant of SEQ ID NO: 50 comprises a sequence with at least 60% identity to SEQ ID NO: 50.
  • the variant of SEQ ID NO: 50 comprises a sequence with at least 70% identity to SEQ ID NO: 50.
  • the variant of SEQ ID NO: 50 comprises a sequence with at least 80% identity to SEQ ID NO: 50. In an embodiment, the variant of SEQ ID NO: 50 comprises a sequence with at least 90% identity to SEQ ID NO:50. In an embodiment, the variant of SEQ ID NO:50 comprises a sequence with at least 95% identity to SEQ ID NO:50. In an embodiment, the variant of SEQ ID NO:50 comprises a sequence with at least 96% identity to SEQ ID NO:50. In an embodiment, the variant of SEQ ID NO:50 comprises a sequence with at least 97% identity to SEQ ID NO:50. In an embodiment, the variant of SEQ ID NO:50 comprises a sequence with at least 98% identity to SEQ ID NO:50.
  • the variant of SEQ ID NO:50 comprises a sequence with at least 99% identity to SEQ ID NO:50.
  • the variant of SEQ ID NO:50 comprises a uridine content of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%.
  • the variant of SEQ ID NO:50 comprises a uridine content of at least 5%.
  • the variant of SEQ ID NO:50 comprises a uridine content of at least 10%.
  • the variant of SEQ ID NO:50 comprises a uridine content of at least 20%.
  • the variant of SEQ ID NO:50 comprises a uridine content of at least 30%.
  • the variant of SEQ ID NO:50 comprises a uridine content of at least 40%. In an embodiment, the variant of SEQ ID NO:50 comprises a uridine content of at least 50%. In an embodiment, the variant of SEQ ID NO:50 comprises a uridine content of at least 60%. In an embodiment, the variant of SEQ ID NO:50 comprises a uridine content of at least 70%. In an embodiment, the variant of SEQ ID NO:50 comprises a uridine content of at least 80%. [00244] In an embodiment, the variant of SEQ ID NO:50 comprises at least 2, 3, 4, 5, 6 or 7 consecutive uridines (e.g., a polyuridine tract).
  • the polyuridine tract in the variant of SEQ ID NO:50 comprises at least 1-7, 2-7, 3-7, 4-7, Attorney Docket No.45817-0124WO1 / MTX959.20 5-7, 6-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-6, or 3-5 consecutive uridines.
  • the polyuridine tract in the variant of SEQ ID NO:50 comprises 4 consecutive uridines.
  • the polyuridine tract in the variant of SEQ ID NO:50 comprises 5 consecutive uridines.
  • the variant of SEQ ID NO:50 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 polyuridine tracts.
  • the variant of SEQ ID NO:50 comprises 3 polyuridine tracts. In an embodiment, the variant of SEQ ID NO:50 comprises 4 polyuridine tracts. In an embodiment, the variant of SEQ ID NO:50 comprises 5 polyuridine tracts. [00246] In an embodiment, one or more of the polyuridine tracts are adjacent to a different polyuridine tract. In an embodiment, each of, e.g., all, the polyuridine tracts are adjacent to each other, e.g., all of the polyuridine tracts are contiguous.
  • one or more of the polyuridine tracts are separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 2, 13, 14, 15, 16, 17, 18.19, 20, 30, 40, 50 or 60 nucleotides.
  • each of, e.g., all of, the polyuridine tracts are separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 2, 13, 14, 15, 16, 17, 18.19, 20, 30, 40, 50 or 60 nucleotides.
  • a first polyuridine tract and a second polyuridine tract are adjacent to each other.
  • a subsequent, e.g., third, fourth, fifth, sixth or seventh, eighth, ninth, or tenth, polyuridine tract is separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 2, 13, 14, 15, 16, 17, 18.19, 20, 30, 40, 50 or 60 nucleotides from the first polyuridine tract, the second polyuridine tract, or any one of the subsequent polyuridine tracts.
  • a first polyuridine tract is separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 2, 13, 14, 15, 16, 17, 18.19, 20, 30, 40, 50 or 60 nucleotides from a subsequent polyuridine tract, e.g., a second, third, fourth, fifth, sixth or seventh, eighth, ninth, or tenth polyuridine tract.
  • a subsequent polyuridine tract e.g., a second, third, fourth, fifth, sixth or seventh, eighth, ninth, or tenth polyuridine tract.
  • one or more of the subsequent polyuridine tracts are adjacent to a different polyuridine tract.
  • the 5′ UTR comprises a Kozak sequence, e.g., a GCCRCC nucleotide sequence, wherein R is an adenine or guanine.
  • the Kozak sequence is disposed at the 3′ end of the 5′UTR sequence.
  • the polynucleotide e.g., mRNA
  • the LNP composition comprises: (i) an ionizable lipid, e.g., an amino lipid; (ii) a sterol or other structural lipid; (iii) a non-cationic helper lipid or phospholipid; and (iv) a PEG-lipid.
  • the LNP compositions of the disclosure are used in a method of expressing a Snu13 polypeptide in a subject.
  • the LNP compositions of the disclosure are used in a method of regulating expression of a polypeptide (e.g., a polypeptide encoded by a polynucleotide comprising a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500)).
  • an LNP composition comprising a polynucleotide disclosed herein encoding a Snu13polypeptide, e.g., as described herein, can be administered with an additional agent, e.g., as described herein.
  • the additional agent is a second polynucleotide comprising (a) a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500) and (b) an open reading frame encoding a polypeptide.
  • 3′UTR sequences have been shown to influence translation, half-life, and subcellular localization of mRNAs (Mayr C., Cold Spring Harb Persp Biol 2019 Oct 1;11(10):a034728).
  • a polynucleotide e.g., mRNA, comprising an open reading frame encoding a Snu13 polypeptide, which polynucleotide has a 3′ UTR that confers an increased half-life, increased expression and/or increased activity of the polypeptide encoded by said polynucleotide, or of the polynucleotide itself.
  • a polynucleotide disclosed herein comprises: (a) a 5′-UTR (e.g., as described herein); (b) a coding region comprising a stop element (e.g., as described herein); and (c) a 3′-UTR (e.g., as provided in Table 5 or a variant or fragment thereof), and LNP compositions comprising the same.
  • the polynucleotide comprises a 3′-UTR comprising a sequence provided in Table 5 or a variant or fragment thereof.
  • the polynucleotide having a 3′ UTR sequence provided in Table 5 or a variant or fragment thereof results in an increased half-life of the polynucleotide, e.g., about 1.5-10-fold increase in half-life of the polynucleotide.
  • the increase in half-life is about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold, or more.
  • the increase in half-life is about 1.5-fold or more.
  • the increase in half-life is about 2-fold or more.
  • the increase in half-life is about 3-fold or more.
  • the increase in half- life is about 4-fold or more. In an embodiment, the increase in half-life is about 5-fold or more. In an embodiment, the increase in half-life is about 6-fold or more. In an embodiment, the increase in half-life is about 7-fold or more. In an embodiment, the increase in half-life is about 8-fold. In an embodiment, the increase in half-life is about 9-fold or more. In an embodiment, the increase in half-life is about 10-fold or more. [00259] In an embodiment, the polynucleotide having a 3′ UTR sequence provided in Table 5 or a variant or fragment thereof, results in a polynucleotide with a mean half-life score of greater than 10.
  • the polynucleotide having a 3′ UTR sequence provided in Table 5 or a variant or fragment thereof results in an increased level and/or activity, e.g., output, of the polypeptide encoded by the polynucleotide.
  • the increase is compared to an otherwise similar polynucleotide which does not have a 3′ UTR, has a different 3′ UTR, or does not have a 3′ UTR of Table 5 or a variant or fragment thereof.
  • the polynucleotide comprises a 3′ UTR sequence provided in Table 5 or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a 3′ UTR sequence provided in Table 5, or a fragment thereof.
  • the 3′ UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, or SEQ ID NO:115.
  • the 3′ UTR comprises the sequence of SEQ ID NO: 100, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 100.
  • the 3′ UTR comprises the sequence of SEQ ID NO: 101, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 101.
  • the 3′ UTR comprises the sequence of SEQ ID NO: 102, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 102.
  • the 3′ UTR comprises the sequence of SEQ ID NO: 103, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 103.
  • the 3′ UTR comprises the sequence of SEQ ID NO: 104, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 104.
  • the 3′ UTR comprises the sequence of SEQ ID NO: 105, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 105.
  • the 3′ UTR comprises the sequence of SEQ ID NO: 106, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 106.
  • the 3′ UTR comprises the sequence of SEQ ID NO: 107, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 107.
  • the 3′ UTR comprises the sequence of SEQ ID NO: 108, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 108.
  • the 3′ UTR comprises the sequence of SEQ ID NO: 109, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 109.
  • the 3′ UTR comprises the sequence of SEQ ID NO: 110, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 110.
  • the 3′ UTR comprises the sequence of SEQ ID NO: 111, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 111.
  • the 3′ UTR comprises the sequence of SEQ ID NO: 112, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 112.
  • the 3′ UTR comprises the sequence of SEQ ID NO: 113, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 113.
  • the 3′ UTR Attorney Docket No.45817-0124WO1 / MTX959.20 comprises the sequence of SEQ ID NO: 114, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 114.
  • the 3′ UTR comprises the sequence of SEQ ID NO: 115, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 115.
  • the 3′ UTR comprises a miRNA binding site of SEQ ID NO: 212, SEQ ID NO: 174, SEQ ID NO: 152 or a combination thereof.
  • the 3′ UTR comprises a plurality of miRNA binding sites, e.g., 2, 3, 4, 5, 6, 7 or 8 miRNA binding sites.
  • the plurality of miRNA binding sites comprises the same or different miRNA binding sites.
  • a polynucleotide encoding a polypeptide wherein the polynucleotide comprises: (a) a 5′-UTR, e.g., as described herein; (b) a coding region comprising a stop element (e.g., as described herein); and (c) a 3′-UTR (e.g., as described herein).
  • the polynucleotide (e.g., mRNA) comprising an open reading frame encoding a Snu13 polypeptide and comprising a 3′ UTR sequence disclosed herein is formulated as an LNP.
  • the LNP composition comprises: Attorney Docket No.45817-0124WO1 / MTX959.20 (i) an ionizable lipid, e.g., an amino lipid; (ii) a sterol or other structural lipid; (iii) a non-cationic helper lipid or phospholipid; and (iv) a PEG-lipid.
  • the LNP compositions of the disclosure are used in a method of expressing a Snu13 polypeptide in a subject.
  • the LNP compositions of the disclosure are used in a method of regulating expression of a polypeptide (e.g., a polypeptide encoded by a polynucleotide comprising a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500)).
  • an LNP composition comprising a polynucleotide disclosed herein encoding a Snu13polypeptide, e.g., as described herein, can be administered with an additional agent, e.g., as described herein.
  • the additional agent is a second polynucleotide comprising (a) a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500) and (b) an open reading frame encoding a polypeptide. 11.
  • Polynucleotides of the invention can include regulatory elements, for example, microRNA (miRNA) binding sites, transcription factor binding sites, structured mRNA sequences and/or motifs, artificial binding sites engineered to act as pseudo-receptors for endogenous nucleic acid binding molecules, and combinations thereof.
  • regulatory elements for example, microRNA (miRNA) binding sites, transcription factor binding sites, structured mRNA sequences and/or motifs, artificial binding sites engineered to act as pseudo-receptors for endogenous nucleic acid binding molecules, and combinations thereof.
  • polynucleotides including such regulatory elements are referred to as including “sensor sequences”.
  • a polynucleotide e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • ORF open reading frame
  • miRNA binding site(s) provides for regulation of polynucleotides of the invention, and in turn, of the polypeptides encoded therefrom, based on tissue-specific and/or cell-type specific expression of naturally-occurring miRNAs.
  • compositions and formulations that comprise any of the polynucleotides described above.
  • the composition or formulation further comprises a delivery agent.
  • the composition or formulation can contain a polynucleotide comprising a sequence optimized nucleic acid sequence disclosed herein which encodes a polypeptide.
  • the composition or formulation can contain a polynucleotide (e.g., a RNA, e.g., an mRNA) comprising a polynucleotide (e.g., an ORF) having significant sequence identity to a sequence optimized nucleic acid sequence disclosed herein which encodes a polypeptide.
  • a polynucleotide e.g., a RNA, e.g., an mRNA
  • an ORF e.g., an ORF having significant sequence identity to a sequence optimized nucleic acid sequence disclosed herein which encodes a polypeptide.
  • the polynucleotide further comprises a miRNA binding site, e.g., a miRNA binding site that binds [00275]
  • a miRNA e.g., a natural-occurring miRNA, is a 19-25 nucleotide long noncoding RNA that binds to a polynucleotide and down-regulates gene expression either by reducing stability or by inhibiting translation of the polynucleotide.
  • a miRNA sequence comprises a “seed” region, i.e., a sequence in the region of positions 2-8 of the mature miRNA.
  • a miRNA seed can comprise positions 2-8 or 2- 7 of the mature miRNA.
  • microRNAs derive enzymatically from regions of RNA transcripts that fold back on themselves to form short hairpin structures often termed a pre-miRNA (precursor-miRNA).
  • a pre-miRNA typically has a two-nucleotide overhang at its 3′ end, and has 3′ hydroxyl and 5′ phosphate groups.
  • This precursor-mRNA is processed in the nucleus and subsequently transported to the cytoplasm where it is further processed by DICER (a RNase III enzyme), to form a mature microRNA of approximately 22 nucleotides.
  • DICER a RNase III enzyme
  • the mature microRNA is then incorporated into a ribonuclear particle to form the RNA-induced silencing complex, RISC, which mediates gene silencing.
  • a miR referred to by number herein can refer to either of the two mature microRNAs originating from opposite arms of the same pre-miRNA (e.g., either the 3p or 5p Attorney Docket No.45817-0124WO1 / MTX959.20 microRNA).
  • miRNA binding site refers to a sequence within a polynucleotide, e.g., within a DNA or within an RNA transcript, including in the 5′UTR and/or 3′UTR, that has sufficient complementarity to all or a region of a miRNA to interact with, associate with or bind to the miRNA.
  • a polynucleotide of the invention comprising an ORF encoding a polypeptide of interest and further comprises one or more miRNA binding site(s).
  • a 5′ UTR and/or 3′ UTR of the polynucleotide comprises the one or more miRNA binding site(s).
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • a miRNA binding site having sufficient complementarity to a miRNA refers to a degree of complementarity sufficient to facilitate miRNA-mediated regulation of a polynucleotide, e.g., miRNA-mediated translational repression or degradation of the polynucleotide.
  • a miRNA binding site having sufficient complementarity to the miRNA refers to a degree of complementarity sufficient to facilitate miRNA-mediated degradation of the polynucleotide, e.g., miRNA-guided RNA-induced silencing complex (RISC)- mediated cleavage of mRNA.
  • the miRNA binding site can have complementarity to, for example, a 19-25 nucleotide long miRNA sequence, to a 19-23 nucleotide long miRNA sequence, or to a 22 nucleotide long miRNA sequence.
  • a miRNA binding site can be complementary to only a portion of a miRNA, e.g., to a portion less than 1, 2, 3, or 4 nucleotides of the full length of a naturally-occurring miRNA sequence, or to a portion less than 1, 2, 3, or 4 nucleotides shorter than a naturally-occurring miRNA sequence.
  • Full or complete complementarity e.g., full complementarity or complete complementarity over all or a significant portion of the length of a naturally- occurring miRNA
  • a miRNA binding site includes a sequence that has complementarity (e.g., partial or complete complementarity) with an miRNA seed sequence.
  • the miRNA binding site includes a sequence that has complete complementarity with a miRNA seed sequence. In some embodiments, a miRNA binding site includes a sequence that has complementarity (e.g., partial or Attorney Docket No.45817-0124WO1 / MTX959.20 complete complementarity) with an miRNA sequence. In some embodiments, the miRNA binding site includes a sequence that has complete complementarity with a miRNA sequence. In other embodiments, the sequence is not completely complementary. In some embodiments, a miRNA binding site has complete complementarity with a miRNA sequence but for 1, 2, or 3 nucleotide substitutions, terminal additions, and/or truncations.
  • the miRNA binding site is the same length as the corresponding miRNA. In other embodiments, the miRNA binding site is one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve nucleotide(s) shorter than the corresponding miRNA at the 5′ terminus, the 3′ terminus, or both. In still other embodiments, the microRNA binding site is two nucleotides shorter than the corresponding microRNA at the 5′ terminus, the 3′ terminus, or both. The miRNA binding sites that are shorter than the corresponding miRNAs are still capable of degrading the mRNA incorporating one or more of the miRNA binding sites or preventing the mRNA from translation.
  • the miRNA binding site binds the corresponding mature miRNA that is part of an active RISC containing Dicer. In another embodiment, binding of the miRNA binding site to the corresponding miRNA in RISC degrades the mRNA containing the miRNA binding site or prevents the mRNA from being translated. In some embodiments, the miRNA binding site has sufficient complementarity to miRNA so that a RISC complex comprising the miRNA cleaves the polynucleotide comprising the miRNA binding site. In other embodiments, the miRNA binding site has imperfect complementarity so that a RISC complex comprising the miRNA induces instability in the polynucleotide comprising the miRNA binding site.
  • the miRNA binding site has imperfect complementarity so that a RISC complex comprising the miRNA represses transcription of the polynucleotide comprising the miRNA binding site.
  • the miRNA binding site has one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve mismatch(es) from the corresponding miRNA.
  • the miRNA binding site has at least about ten, at least about eleven, at least about twelve, at least about thirteen, at least about fourteen, Attorney Docket No.45817-0124WO1 / MTX959.20 at least about fifteen, at least about sixteen, at least about seventeen, at least about eighteen, at least about nineteen, at least about twenty, or at least about twenty-one contiguous nucleotides complementary to at least about ten, at least about eleven, at least about twelve, at least about thirteen, at least about fourteen, at least about fifteen, at least about sixteen, at least about seventeen, at least about eighteen, at least about nineteen, at least about twenty, or at least about twenty-one, respectively, contiguous nucleotides of the corresponding miRNA.
  • the polynucleotide By engineering one or more miRNA binding sites into a polynucleotide of the invention, the polynucleotide can be targeted for degradation or reduced translation, provided the miRNA in question is available. This can reduce off-target effects upon delivery of the polynucleotide. For example, if a polynucleotide of the invention is not intended to be delivered to a tissue or cell but ends up is said tissue or cell, then a miRNA abundant in the tissue or cell can inhibit the expression of the gene of interest if one or multiple binding sites of the miRNA are engineered into the 5′ UTR and/or 3′ UTR of the polynucleotide.
  • incorporation of one or more miRNA binding sites into an mRNA of the disclosure may reduce the hazard of off-target effects upon nucleic acid molecule delivery and/or enable tissue-specific regulation of expression of a polypeptide encoded by the mRNA.
  • incorporation of one or more miRNA binding sites into an mRNA of the disclosure can modulate immune responses upon nucleic acid delivery in vivo.
  • incorporation of one or more miRNA binding sites into an mRNA of the disclosure can modulate accelerated blood clearance (ABC) of lipid-comprising compounds and compositions described herein.
  • ABS accelerated blood clearance
  • miRNA binding sites can be removed from polynucleotide sequences in which they naturally occur to increase protein expression in specific tissues.
  • a binding site for a specific miRNA can be removed from a polynucleotide to improve protein expression in tissues or cells containing the miRNA.
  • Regulation of expression in multiple tissues can be accomplished through introduction or removal of one or more miRNA binding sites, e.g., one or more distinct miRNA binding sites. The decision whether to remove or insert a miRNA binding site can be made based on miRNA expression patterns and/or their profilings Attorney Docket No.45817-0124WO1 / MTX959.20 in tissues and/or cells in development and/or disease.
  • tissues where miRNA are known to regulate mRNA, and thereby protein expression include, but are not limited to, liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-1d, miR-149), kidney (miR-192, miR-194, miR- 204), and lung epithelial cells (let-7, miR-133, miR-126).
  • liver miR-122
  • muscle miR-133, miR-206, miR-208
  • endothelial cells miR-17-92, miR-126
  • myeloid cells miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, mi
  • miRNAs are known to be differentially expressed in immune cells (also called hematopoietic cells), such as antigen presenting cells (APCs) (e.g., dendritic cells and macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes, granulocytes, natural killer cells, etc.
  • APCs antigen presenting cells
  • Immune cell specific miRNAs are involved in immunogenicity, autoimmunity, the immune-response to infection, inflammation, as well as unwanted immune response after gene therapy and tissue/organ transplantation. Immune cells specific miRNAs also regulate many aspects of development, proliferation, differentiation and apoptosis of hematopoietic cells (immune cells).
  • miR-142 and miR-146 are exclusively expressed in immune cells, particularly abundant in myeloid dendritic cells. It has been demonstrated that the immune response to a polynucleotide can be shut-off by adding miR-142 binding sites to the 3′-UTR of the polynucleotide, enabling more stable gene transfer in tissues and cells.
  • miR-142 efficiently degrades exogenous polynucleotides in antigen presenting cells and suppresses cytotoxic elimination of transduced cells (e.g., Annoni A et al., blood, 2009, 114, 5152-5161; Brown BD, et al., Nat med.2006, 12(5), 585-591; Brown BD, et al., blood, 2007, 110(13): 4144-4152, each of which is incorporated herein by reference in its entirety).
  • An antigen-mediated immune response can refer to an immune response triggered by foreign antigens, which, when entering an organism, are processed by the antigen presenting cells and displayed on the surface of the antigen presenting cells. T cells can recognize the presented antigen and induce a cytotoxic elimination of cells that express the antigen.
  • Introducing one or more (e.g., one, two, or three) miR-142 binding sites into the 5′ UTR and/or 3′UTR of a polynucleotide of the invention can selectively repress gene expression in antigen presenting cells through miR-142 mediated degradation, limiting antigen presentation in antigen presenting cells (e.g., dendritic cells) and thereby preventing antigen-mediated immune response after the delivery of the polynucleotide.
  • the polynucleotide is then stably expressed in target tissues or cells without triggering cytotoxic elimination.
  • polynucleotides of the invention contain two or more (e.g., two, three, four or more) miR bindings sites from: (i) the group consisting of miR-142, miR-144, miR-150, miR-155 and miR-223 (which are expressed in many hematopoietic cells); or (ii) the group consisting of miR-142, miR150, miR-16 and miR-223 (which are expressed in B cells); or the group consisting of miR-223, miR-451, miR-26a, miR-16 (which are expressed in progenitor hematopoietic cells).
  • miR-142, miR-144, miR-150, miR-155 and miR-223 which are expressed in many hematopoietic cells
  • miR-142, miR150, miR-16 and miR-223 which are expressed in B cells
  • miR-223, miR-451, miR-26a, miR-16 which are expressed in progenitor hema
  • miR-142 and miR-126 may also be beneficial to combine various miRs such that multiple cell types of interest are targeted at the same time (e.g., miR-142 and miR-126 to target many cells of the hematopoietic lineage and endothelial cells).
  • polynucleotides of the invention comprise two or more (e.g., two, three, four or more) miRNA bindings sites, wherein: (i) at least one of the miRs targets cells of the hematopoietic lineage (e.g., miR-142, miR- 144, miR-150, miR-155 or miR-223) and at least one of the miRs targets plasmacytoid dendritic cells, platelets or endothelial cells (e.g., miR-126); or (ii) at least one of the miRs targets B cells (e.g., miR-142, miR150, miR-16 or miR-223) and at least one of the miRs targets plasmacytoid dendritic cells, platelets or Attorney Docket No.45817-0124WO1 / MTX959.20 endothelial cells (e.g., miR-126); or (iii) at least one of the miR
  • polynucleotides of the present invention can comprise one or more miRNA binding sequences that bind to one or more miRs that are expressed in conventional immune cells or any cell that expresses TLR7 and/or TLR8 and secrete pro-inflammatory cytokines and/or chemokines (e.g., in immune cells of peripheral lymphoid organs and/or splenocytes and/or endothelial cells).
  • miRNA binding sequences that bind to one or more miRs that are expressed in conventional immune cells or any cell that expresses TLR7 and/or TLR8 and secrete pro-inflammatory cytokines and/or chemokines (e.g., in immune cells of peripheral lymphoid organs and/or splenocytes and/or endothelial cells).
  • incorporation into an mRNA of one or more miRs that are expressed in conventional immune cells or any cell that expresses TLR7 and/or TLR8 and secrete pro-inflammatory cytokines and/or chemokines reduces or inhibits immune cell activation (e.g., B cell activation, as measured by frequency of activated B cells) and/or cytokine production (e.g., production of IL-6, IFN- ⁇ and/or TNF ⁇ ).
  • incorporation into an mRNA of one or more miRs that are expressed in conventional immune cells or any cell that expresses TLR7 and/or TLR8 and secrete pro-inflammatory cytokines and/or chemokines can reduce or inhibit an anti-drug antibody (ADA) response against a protein of interest encoded by the mRNA.
  • ADA anti-drug antibody
  • polynucleotides of the invention can comprise one or more miR binding sequences Attorney Docket No.45817-0124WO1 / MTX959.20 that bind to one or more miRNAs expressed in conventional immune cells or any cell that expresses TLR7 and/or TLR8 and secrete pro-inflammatory cytokines and/or chemokines (e.g., in immune cells of peripheral lymphoid organs and/or splenocytes and/or endothelial cells).
  • miR binding sequences Attorney Docket No.45817-0124WO1 / MTX959.20 that bind to one or more miRNAs expressed in conventional immune cells or any cell that expresses TLR7 and/or TLR8 and secrete pro-inflammatory cytokines and/or chemokines (e.g., in immune cells of peripheral lymphoid organs and/or splenocytes and/or endothelial cells).
  • incorporation into an mRNA of one or more miR binding sites reduces or inhibits accelerated blood clearance (ABC) of the lipid-comprising compound or composition for use in delivering the mRNA. Furthermore, it has now been discovered that incorporation of one or more miR binding sites into an mRNA reduces serum levels of anti-PEG anti- IgM (e.g., reduces or inhibits the acute production of IgMs that recognize polyethylene glycol (PEG) by B cells) and/or reduces or inhibits proliferation and/or activation of plasmacytoid dendritic cells following administration of a lipid- comprising compound or composition comprising the mRNA.
  • APC accelerated blood clearance
  • miR sequences may correspond to any known microRNA expressed in immune cells, including but not limited to those taught in US Publication US2005/0261218 and US Publication US2005/0059005, the contents of which are incorporated herein by reference in their entirety.
  • Non-limiting examples of miRs expressed in immune cells include those expressed in spleen cells, myeloid cells, dendritic cells, plasmacytoid dendritic cells, B cells, T cells and/or macrophages.
  • miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24 and miR-27 are expressed in myeloid cells
  • miR-155 is expressed in dendritic cells
  • miR-146 is upregulated in macrophages upon TLR stimulation
  • miR-126 is expressed in plasmacytoid dendritic cells.
  • the miR(s) is expressed abundantly or preferentially in immune cells.
  • miR-142 miR-142-3p and/or miR-142-5p
  • miR-126 miR-126-3p and/or miR-126-5p
  • miR-146 miR-146-3p and/or miR-146-5p
  • miR-155 miR- 155-3p and/or miR155-5p
  • the polynucleotide of the invention comprises three copies of the same miRNA binding site.
  • the polynucleotide of the invention comprises two or more (e.g., two, three, four) copies of at least two different miR binding sites expressed in immune cells.
  • the polynucleotide of the invention comprises at least two miR binding sites for microRNAs expressed in immune cells, wherein one of the miR binding sites is for miR-142-3p.
  • the polynucleotide of the invention comprises binding sites for miR-142-3p and miR-155 (miR-155-3p or miR-155-5p), miR-142-3p and miR-146 (miR-146-3 or miR-146-5p), or miR-142-3p and miR-126 (miR-126-3p or miR-126-5p). [00299] In another embodiment, the polynucleotide of the invention comprises at least two miR binding sites for microRNAs expressed in immune cells, wherein one of the miR binding sites is for miR-126-3p.
  • the polynucleotide of the invention comprises binding sites for miR-126-3p and miR-155 (miR-155-3p or miR-155-5p), miR-126-3p and miR-146 (miR-146-3p or miR-146- 5p), or miR-126-3p and miR-142 (miR-142-3p or miR-142-5p).
  • the polynucleotide of the invention comprises at least two miR binding sites for microRNAs expressed in immune cells, wherein one of the miR binding sites is for miR-142-5p.
  • the polynucleotide of the invention comprises binding sites for miR-142-5p and miR-155 (miR-155-3p or miR-155-5p), miR-142-5p and miR-146 (miR-146-3 or miR-146-5p), or miR-142-5p and miR-126 (miR-126-3p or miR-126-5p).
  • the polynucleotide of the invention comprises at least two miR binding sites for microRNAs expressed in immune cells, wherein one of the miR binding sites is for miR-155-5p.
  • the polynucleotide of the invention comprises binding sites for miR-155-5p and miR-142 (miR-142-3p or miR-142-5p), miR-155-5p and miR-146 (miR-146-3 or miR-146-5p), or miR-155-5p and miR-126 (miR-126-3p or miR-126-5p).
  • a polynucleotide of the invention comprises a miRNA binding site, wherein the miRNA binding site comprises one or more nucleotide sequences selected from Table 6, including one or more copies of any one Attorney Docket No.45817-0124WO1 / MTX959.20 or more of the miRNA binding site sequences.
  • a polynucleotide of the invention further comprises at least one, two, three, four, five, six, seven, eight, nine, ten, or more of the same or different miRNA binding sites selected from Table 6, including any combination thereof.
  • the miRNA binding site binds to miR-142 or is complementary to miR-142.
  • the miR-142 comprises SEQ ID NO:172.
  • the miRNA binding site binds to miR-142-3p or miR-142-5p.
  • the miR-142-3p binding site comprises SEQ ID NO:174.
  • the miR-142-5p binding site comprises SEQ ID NO:210.
  • the miRNA binding site comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:174 or SEQ ID NO:210.
  • the miRNA binding site binds to miR-126 or is complementary to miR-126.
  • the miR-126 comprises SEQ ID NO: 150.
  • the miRNA binding site binds to miR-126-3p or miR-126-5p.
  • the miR-126-3p binding site comprises SEQ ID NO: 152.
  • the miR-126-5p binding site comprises SEQ ID NO: 154.
  • the miRNA binding site comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 152 or SEQ ID NO: 154.
  • the 3′ UTR comprises two miRNA binding sites, wherein a first miRNA binding site binds to miR-142 and a second miRNA binding site binds to miR-126.
  • Table 6. miR-142, miR-126, and miR-142 and miR-126 binding sites SEQ ID N O Description Sequence Attorney Docket No.45817-0124WO1 / MTX959.20 SEQ ID N O.
  • the insertion site in the polynucleotide can be anywhere in the polynucleotide as long as the insertion of the miRNA binding site in the polynucleotide does not interfere with the translation of a functional polypeptide in the absence of the corresponding miRNA; and in the presence of the miRNA, the insertion of the miRNA binding site in the polynucleotide and the binding of the miRNA binding site to the corresponding miRNA are capable of degrading the polynucleotide or preventing the translation of the polynucleotide.
  • a miRNA binding site is inserted in at least about 30 nucleotides downstream from the stop codon of an ORF in a polynucleotide of the invention comprising the ORF.
  • a miRNA binding site is inserted in at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, or at
  • a miRNA binding site is inserted in about 10 nucleotides to about 100 nucleotides, about 20 nucleotides to about 90 nucleotides, about 30 nucleotides to Attorney Docket No.45817-0124WO1 / MTX959.20 about 80 nucleotides, about 40 nucleotides to about 70 nucleotides, about 50 nucleotides to about 60 nucleotides, about 45 nucleotides to about 65 nucleotides downstream from the stop codon of an ORF in a polynucleotide of the invention.
  • a miRNA binding site is inserted within the 3′ UTR immediately following the stop codon of the coding region within the polynucleotide of the invention, e.g., mRNA. In some embodiments, if there are multiple copies of a stop codon in the construct, a miRNA binding site is inserted immediately following the final stop codon. In some embodiments, a miRNA binding site is inserted further downstream of the stop codon, in which case there are 3′ UTR bases between the stop codon and the miR binding site(s).
  • a codon optimized open reading frame encoding a polypeptide of interest comprises a stop codon and the at least one microRNA binding site is located within the 3′ UTR 1-100 nucleotides after the stop codon.
  • the codon optimized open reading frame encoding the polypeptide of interest comprises a stop codon and the at least one microRNA binding site for a miR expressed in immune cells is located within the 3′ UTR 30-50 nucleotides after the stop codon.
  • the codon optimized open reading frame encoding the polypeptide of interest comprises a stop codon and the at least one microRNA binding site for a miR expressed in immune cells is located within the 3′ UTR at least 50 nucleotides after the stop codon.
  • the codon optimized open reading frame encoding the polypeptide of interest comprises a stop codon and the at least one microRNA binding site for a miR expressed in immune cells is located within the 3′ UTR immediately after the stop codon, or within the 3′ UTR 15-20 nucleotides after the stop codon or within the 3′ UTR 70-80 nucleotides after the stop codon.
  • the 3′ UTR comprises more than one miRNA binding site (e.g., 2-4 miRNA binding sites), wherein there can be a spacer region (e.g., of 10-100, 20-70 or 30-50 nucleotides in length) between each miRNA binding site.
  • the 3′ UTR comprises a spacer region between the end of the miRNA binding site(s) and the poly A tail nucleotides.
  • a spacer region of 10-100, 20-70 or 30-50 nucleotides in length can be situated between the end of the miRNA binding site(s) and the beginning of the poly A tail.
  • the 3′ UTR comprises more than one stop codon, wherein at least one miRNA binding site is positioned downstream of the stop codons.
  • a 3′ UTR can comprise 1, 2 or 3 stop codons.
  • triple stop codons include: UGAUAAUAG, UGAUAGUAA, UAAUGAUAG, UGAUAAUAA, UGAUAGUAG, UAAUGAUGA, UAAUAGUAG, UGAUGAUGA, UAAUAAUAA, and UAGUAGUAG.
  • a 3′ UTR for example, 1, 2, 3 or 4 miRNA binding sites, e.g., miR-142-3p binding sites, can be positioned immediately adjacent to the stop codon(s) or at any number of nucleotides downstream of the final stop codon.
  • these binding sites can be positioned directly next to each other in the construct (i.e., one after the other) or, alternatively, spacer nucleotides can be positioned between each binding site.
  • the 3′ UTR comprises three stop codons with a single miR-142-3p binding site located downstream of the 3rd stop codon.
  • the polynucleotide of the invention comprises a 5′ UTR comprising the nucleotide sequence of SEQ ID NO:50, a codon optimized open reading frame encoding Snu13, a 3′ UTR comprising the at least one miRNA binding site for a miR expressed in immune cells, and a 3′ tailing region of linked nucleosides.
  • the 3′ UTR comprises 1-4, at least two, one, two, three or four miRNA binding sites for miRs expressed in immune cells, preferably abundantly or preferentially expressed in immune cells.
  • the at least one miRNA expressed in immune cells is a miR-142-3p microRNA binding site.
  • the miR-142-3p microRNA binding site comprises the sequence shown in SEQ ID NO: 174.
  • the at least one miRNA expressed in immune cells is a miR-126 microRNA binding site.
  • the miR-126 binding site is a miR-126-3p binding site.
  • the miR-126-3p microRNA binding site comprises the sequence shown in SEQ ID NO: 152.
  • Non-limiting exemplary sequences for miRs to which a microRNA binding site(s) of the disclosure can bind include the following: miR-142-3p (SEQ ID NO: 173), miR-142-5p (SEQ ID NO: 175), miR-146-3p (CCUCUGAAAUUCAGUUCUUCAG; SEQ ID NO: 155), miR-146-5p Attorney Docket No.45817-0124WO1 / MTX959.20 (UGAGAACUGAAUUCCAUGGGUU; SEQ ID NO: 156), miR-155-3p (CUCCUACAUAUUAGCAUUAACA; SEQ ID NO: 157), miR-155-5p (UUAAUGCUAAUCGUGAUAGGGGU; SEQ ID NO: 158), miR-126-3p (SEQ ID NO: 151), miR-126-5p (SEQ ID NO: 153), miR-16-3p (CCAGUAUUAACUGUGCUGCUGA; SEQ ID NO: 159), miR
  • miR sequences expressed in immune cells are known and available in the art, for example at the University of Manchester’s microRNA database, miRBase. Sites that bind any of the aforementioned miRs can be designed based on Watson-Crick complementarity to the miR, typically 100% complementarity to the miR, and inserted into an mRNA construct of the disclosure as described herein.
  • a polynucleotide of the present invention (e.g., and mRNA, e.g., the 3′ UTR thereof) can comprise at least one miRNA binding site to thereby reduce or inhibit accelerated blood clearance, for example by reducing or inhibiting production of IgMs, e.g., against PEG, by B cells and/or reducing or inhibiting proliferation and/or activation of pDCs, and can comprise at least one miRNA binding site for modulating tissue expression of an encoded protein of interest.
  • miRNA gene regulation can be influenced by the sequence surrounding the miRNA such as, but not limited to, the species of the surrounding sequence, the type of sequence (e.g., heterologous, homologous, exogenous, endogenous, or artificial), regulatory elements in the surrounding sequence and/or structural elements in the surrounding sequence.
  • the miRNA can be influenced by the 5′UTR and/or 3′UTR.
  • Attorney Docket No.45817-0124WO1 / MTX959.20 As a non-limiting example, a non-human 3′UTR can increase the regulatory effect of the miRNA sequence on the expression of a polypeptide of interest compared to a human 3′ UTR of the same sequence type.
  • other regulatory elements and/or structural elements of the 5′ UTR can influence miRNA mediated gene regulation.
  • a regulatory element and/or structural element is a structured IRES (Internal Ribosome Entry Site) in the 5′ UTR, which is necessary for the binding of translational elongation factors to initiate protein translation. EIF4A2 binding to this secondarily structured element in the 5′-UTR is necessary for miRNA mediated gene expression (Meijer HA et al., Science, 2013, 340, 82-85, herein incorporated by reference in its entirety).
  • the polynucleotides of the invention can further include this structured 5′ UTR in order to enhance microRNA mediated gene regulation.
  • At least one miRNA binding site can be engineered into the 3′ UTR of a polynucleotide of the invention.
  • at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more miRNA binding sites can be engineered into a 3′ UTR of a polynucleotide of the invention.
  • 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2, or 1 miRNA binding sites can be engineered into the 3′UTR of a polynucleotide of the invention.
  • miRNA binding sites incorporated into a polynucleotide of the invention can be the same or can be different miRNA sites.
  • a combination of different miRNA binding sites incorporated into a polynucleotide of the invention can include combinations in which more than one copy of any of the different miRNA sites are incorporated.
  • miRNA binding sites incorporated into a polynucleotide of the invention can target the same or different tissues in the body.
  • a miRNA binding site can be engineered near the 5′ terminus of the 3′UTR, about halfway between the 5′ terminus and 3′ terminus of the 3′UTR and/or near the 3′ terminus of the 3′ UTR in a polynucleotide of the invention.
  • a miRNA binding site can be engineered near the 5′ Attorney Docket No.45817-0124WO1 / MTX959.20 terminus of the 3′UTR and about halfway between the 5′ terminus and 3′ terminus of the 3′UTR.
  • a miRNA binding site can be engineered near the 3′ terminus of the 3′UTR and about halfway between the 5′ terminus and 3′ terminus of the 3′ UTR.
  • a miRNA binding site can be engineered near the 5′ terminus of the 3′ UTR and near the 3′ terminus of the 3′ UTR.
  • a 3′UTR can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNA binding sites.
  • the miRNA binding sites can be complementary to a miRNA, miRNA seed sequence, and/or miRNA sequences flanking the seed sequence.
  • the expression of a polynucleotide of the invention can be controlled by incorporating at least one sensor sequence in the polynucleotide and Formulating the polynucleotide for administration.
  • a polynucleotide of the invention can be targeted to a tissue or cell by incorporating a miRNA binding site and Formulating the polynucleotide in a lipid nanoparticle comprising an ionizable amino lipid, including any of the lipids described herein.
  • a polynucleotide of the invention can be engineered for more targeted expression in specific tissues, cell types, or biological conditions based on the expression patterns of miRNAs in the different tissues, cell types, or biological conditions. Through introduction of tissue-specific miRNA binding sites, a polynucleotide of the invention can be designed for optimal protein expression in a tissue or cell, or in the context of a biological condition.
  • a polynucleotide of the invention can be designed to incorporate miRNA binding sites that either have 100% identity to known miRNA seed sequences or have less than 100% identity to miRNA seed sequences.
  • a polynucleotide of the invention can be designed to incorporate miRNA binding sites that have at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to known miRNA seed sequences.
  • the miRNA seed sequence can be partially mutated to decrease miRNA binding affinity and as such result in reduced downmodulation of the polynucleotide.
  • a miRNA sequence can be incorporated into the loop of a stem loop.
  • a miRNA seed sequence can be incorporated in the loop of a stem loop and a miRNA binding site can be incorporated into the 5′ or 3′ stem of the stem loop.
  • a polynucleotide of the invention can include at least one miRNA in order to dampen the antigen presentation by antigen presenting cells.
  • the miRNA can be the complete miRNA sequence, the miRNA seed sequence, the miRNA sequence without the seed, or a combination thereof.
  • a miRNA incorporated into a polynucleotide of the invention can be specific to the hematopoietic system.
  • a miRNA incorporated into a polynucleotide of the invention to dampen antigen presentation is miR-142-3p.
  • a polynucleotide of the invention can include at least one miRNA in order to dampen expression of the encoded polypeptide in a tissue or cell of interest.
  • a polynucleotide of the invention can include at least one miR-142-3p binding site, miR-142-3p seed sequence, miR- 142-3p binding site without the seed, miR-142-5p binding site, miR-142-5p seed sequence, miR-142-5p binding site without the seed, miR-146 binding site, miR-146 seed sequence and/or miR-146 binding site without the seed sequence.
  • a polynucleotide of the invention can comprise at least one miRNA binding site in the 3′UTR in order to selectively degrade mRNA therapeutics in the immune cells to subdue unwanted immunogenic reactions caused by therapeutic delivery.
  • the miRNA binding site can make a polynucleotide of the invention more unstable in antigen presenting cells.
  • these miRNAs include miR-142-5p, miR-142-3p, miR- 146a-5p, and miR-146-3p.
  • a polynucleotide of the invention comprises at least one miRNA sequence in a region of the polynucleotide that can interact with a RNA binding protein.
  • the polynucleotide of the invention e.g., a RNA, e.g., an mRNA
  • the polynucleotide of the invention comprising (i) a sequence-optimized nucleotide sequence (e.g., an ORF) encoding a Snu13 polypeptide (e.g., a Snu13 polypeptide described herein or a functional fragment or variant thereof) and (ii) a miRNA binding site (e.g., a miRNA binding site that binds to miR-142) and/or a miRNA binding site that binds to miR- 126.
  • a sequence-optimized nucleotide sequence e.g., an ORF
  • a Snu13 polypeptide e.g., a Sn
  • the disclosure also includes a polynucleotide that comprises both a 5′ Cap and a polynucleotide of the present invention (e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide to be expressed).
  • a polynucleotide that comprises both a 5′ Cap and a polynucleotide of the present invention (e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide to be expressed).
  • CBP mRNA Cap Binding Protein
  • Endogenous mRNA molecules can be 5′-end capped generating a 5′-ppp- 5′-triphosphate linkage between a terminal guanosine cap residue and the 5′-terminal transcribed sense nucleotide of the mRNA molecule. This 5′-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue.
  • the ribose sugars of the terminal and/or anteterminal transcribed nucleotides of the 5′ end of the mRNA can optionally also be 2′-O-methylated.5′-decapping through hydrolysis and cleavage of the guanylate cap structure can target a nucleic acid molecule, such as an mRNA molecule, for degradation.
  • the polynucleotides of the present invention e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide
  • incorporate a cap moiety incorporate a cap moiety.
  • polynucleotides of the present invention comprise a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half- life. Because cap structure hydrolysis requires cleavage of 5′-ppp-5′ phosphorodiester Attorney Docket No.45817-0124WO1 / MTX959.20 linkages, modified nucleotides can be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) can be used with ⁇ -thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5′-ppp-5′ cap.
  • a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) can be used with ⁇ -thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5′-ppp-5
  • Additional modified guanosine nucleotides can be used such as ⁇ -methyl-phosphonate and seleno-phosphate nucleotides.
  • Additional modifications include, but are not limited to, 2′-O-methylation of the ribose sugars of 5′-terminal and/or 5′-anteterminal nucleotides of the polynucleotide (as mentioned above) on the 2′-hydroxyl group of the sugar ring.
  • Multiple distinct 5′-cap structures can be used to generate the 5′-cap of a nucleic acid molecule, such as a polynucleotide that functions as an mRNA molecule.
  • Cap analogs which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wild-type or physiological) 5′-caps in their chemical structure, while retaining cap function.
  • Cap analogs can be chemically (i.e., non-enzymatically) or enzymatically synthesized and/or linked to the polynucleotides of the invention.
  • the Anti-Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5′-5′-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3′-O-methyl group (i.e., N7,3′-O-dimethyl-guanosine-5′- triphosphate-5′-guanosine (m 7 G-3′mppp-G; which can equivalently be designated 3′ O-Me-m 7 G(5′)ppp(5′)G).
  • the 3′-O atom of the other, unmodified, guanine becomes linked to the 5′-terminal nucleotide of the capped polynucleotide.
  • N7- and 3′-O- methlyated guanine provides the terminal moiety of the capped polynucleotide.
  • Another exemplary cap is mCAP, which is similar to ARCA but has a 2′- O-methyl group on guanosine (i.e., N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′- guanosine, m 7 Gm-ppp-G).
  • the cap is m 7 GpppG2 ⁇ OMe or m 7 G-ppp-Gm-A (i.e., N7,guanosine-5′-triphosphate-2′-O-dimethyl-guanosine-adenosine).
  • the cap is a dinucleotide cap analog.
  • the dinucleotide cap analog can be modified at different phosphate positions with a boranophosphate group or a phosphoroselenoate group such as the Attorney Docket No.45817-0124WO1 / MTX959.20 dinucleotide cap analogs described in U.S. Patent No.
  • the cap is a cap analog is a N7-(4- chlorophenoxyethyl) substituted dinucleotide form of a cap analog known in the art and/or described herein.
  • Non-limiting examples of a N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog include a N7-(4-chlorophenoxyethyl)- G(5′)ppp(5′)G and a N7-(4-chlorophenoxyethyl)-m 3′-O G(5′)ppp(5′)G cap analog (See, e.g., the various cap analogs and the methods of synthesizing cap analogs described in Kore et al. Bioorganic & Medicinal Chemistry 201321:4570-4574; the contents of which are herein incorporated by reference in its entirety).
  • a cap analog of the present invention is a 4-chloro/bromophenoxyethyl analog.
  • Polynucleotides of the invention can also be capped post-manufacture (whether IVT or chemical synthesis), using enzymes, in order to generate more authentic 5′-cap structures.
  • the phrase "more authentic" refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature.
  • a "more authentic" feature is better representative of an endogenous, wild-type, natural or physiological cellular function and/or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects.
  • Non-limiting examples of more authentic 5′cap structures of the present invention are those that, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5′ endonucleases and/or reduced 5′decapping, as compared to synthetic 5′cap structures known in the art (or to a wild-type, natural or physiological 5′cap structure).
  • recombinant Vaccinia Virus Capping Enzyme and recombinant 2′-O- methyltransferase enzyme can create a canonical 5′-5′-triphosphate linkage between the 5′-terminal nucleotide of a polynucleotide and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5′-terminal nucleotide of the mRNA contains a 2′-O-methyl.
  • Cap1 structure Such a structure is termed the Cap1 structure.
  • Cap structures include, but are not limited to, Attorney Docket No.45817-0124WO1 / MTX959.20 7mG(5′)ppp(5′)N1pN2p (cap 0), 7mG(5′)ppp(5′)N1mpNp (cap 1), and 7mG(5′)- ppp(5′)N1mpN2mp (cap 2).
  • capping chimeric polynucleotides post- manufacture can be more efficient as nearly 100% of the chimeric polynucleotides can be capped. This is in contrast to ⁇ 80% when a cap analog is linked to a chimeric polynucleotide in the course of an in vitro transcription reaction.
  • 5′ terminal caps can include endogenous caps or cap analogs.
  • a 5′ terminal cap can comprise a guanine analog.
  • guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo- guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.
  • exemplary caps including those that can be used in co-transcriptional capping methods for ribonucleic acid (RNA) synthesis, using RNA polymerase, e.g., wild type RNA polymerase or variants thereof, e.g., such as those variants described herein.
  • caps can be added when RNA is produced in a “one-pot” reaction, without the need for a separate capping reaction.
  • the methods comprise reacting a polynucleotide template with an RNA polymerase variant, nucleoside triphosphates, and a cap analog under in vitro transcription reaction conditions to produce RNA transcript.
  • cap includes the inverted G nucleotide and can comprise one or more additional nucleotides 3’ of the inverted G nucleotide, e.g., 1, 2, 3, or more nucleotides 3’ of the inverted G nucleotide and 5’ to the 5’ UTR, e.g., a 5’ UTR described herein.
  • Exemplary caps comprise a sequence of GG, GA, or GGA, wherein the underlined, italicized G is an in inverted G nucleotide followed by a 5’-5’- triphosphate group.
  • a cap comprises a compound of formula (I) Attorney Docket No.45817-0124WO1 / MTX959.20 a ; a or ring B2 and ring B3 each independently is a nucleobase or a modified nucleobase; X2 is O, S(O)p, NR24 or CR25R26 in which p is 0, 1, or 2; Y 0 is O or CR 6 R 7 ; Y1 is O, S(O)n, CR6R7, or NR8, in which n is 0, 1 , or 2; each --- is a single bond or absent, wherein when each --- is a single bond, Yi is O, S(O)n, CR6R7, or NR8; and when each --- is absent, Y1 is void; Y2 is (OP(O)R4)m in which m is 0, 1, or 2, or -O-(CR40R41)u-Q0-(CR42R43)
  • a cap analog may include any of the cap analogs described in international publication WO 2017/066797, published on 20 April 2017, incorporated by reference herein in its entirety.
  • the B 2 middle position can be a non-ribose molecule, such as arabinose.
  • R2 is ethyl-based.
  • a cap comprises the following structure: Attorney Docket No.45817-0124WO1 / MTX959.20
  • a cap comprises the following structure: .
  • R is a methyl group (e.g., C1 alkyl).
  • R is an ethyl group (e.g., C 2 alkyl).
  • a cap comprises a sequence selected from the following sequences: GAA, GAC, GAG, GAU, GCA, GCC, GCG, GCU, GGA , GGC, GGG, GGU, GUA, GUC, GUG, and GUU.
  • a cap comprises GAA.
  • a cap comprises GAC.
  • a cap comprises GAG. In some embodiments, a cap comprises GAU. In some embodiments, a cap comprises GCA. In some embodiments, a cap comprises GCC. In some embodiments, a cap comprises GCG. In some embodiments, a cap comprises GCU. In some embodiments, a cap comprises GGA. In some embodiments, a cap comprises GGC. In some embodiments, a cap comprises GGG. In some embodiments, a cap comprises GGU. In some embodiments, a cap comprises GUA. In some embodiments, a cap comprises GUC. In some embodiments, a cap comprises GUG. In some embodiments, a cap comprises GUU.
  • a cap comprises a sequence selected from the following sequences: m 7 GpppApA, m 7 GpppApC, m 7 GpppApG, m 7 GpppApU, m 7 GpppCpA, m 7 GpppCpC, m 7 GpppCpG, m 7 GpppCpU, m 7 GpppGpA, m 7 GpppGpC, m 7 GpppGpG, m 7 GpppGpU, m 7 GpppUpA, m 7 GpppUpC, m 7 GpppUpG, and m 7 GpppUpU.
  • a cap comprises m 7 GpppApA. In some embodiments, a cap comprises m 7 GpppApC. In some embodiments, a cap comprises m 7 GpppApG. In some embodiments, a cap comprises m 7 GpppApU. In some embodiments, a cap comprises m 7 GpppCpA. In some embodiments, a cap comprises m 7 GpppCpC. In some embodiments, a cap comprises m 7 GpppCpG. In some embodiments, a cap comprises m 7 GpppCpU. In some embodiments, a cap comprises m 7 GpppGpA.
  • a cap comprises m 7 GpppGpC. In some embodiments, a cap comprises m 7 GpppGpG. In some embodiments, a cap comprises m 7 GpppGpU. In some embodiments, a cap comprises m 7 GpppUpA. In some embodiments, a cap comprises m 7 GpppUpC. In some embodiments, a cap comprises m 7 GpppUpG. In some embodiments, a cap comprises m 7 GpppUpU.
  • a cap in some embodiments, comprises a sequence selected from the following sequences: m 7 G 3 ⁇ OMe pppApA, m 7 G 3 ⁇ OMe pppApC, m 7 G 3 ⁇ OMe pppApG, m 7 G 3 ⁇ OMe pppApU, m 7 G 3 ⁇ OMe pppCpA, m 7 G 3 ⁇ OMe pppCpC, m 7 G 3 ⁇ OMe pppCpG, m 7 G3 ⁇ OMepppCpU, m 7 G3 ⁇ OMepppGpA, m 7 G3 ⁇ OMepppGpC, m 7 G3 ⁇ OMepppGpG, m 7 G3 ⁇ OMepppGpU, m 7 G3 ⁇ OMepppUpA, m 7 G3 ⁇ OMepppUpC, m 7 G3 ⁇ OMepppUpC
  • a cap comprises m 7 G3 ⁇ OMepppApA. In some embodiments, a cap comprises m 7 G 3 ⁇ OMe pppApC. In some embodiments, a cap comprises m 7 G3 ⁇ OMepppApG. In some embodiments, a cap comprises m 7 G3 ⁇ OMepppApU. In some embodiments, a cap comprises m 7 G3 ⁇ OMepppCpA. In some embodiments, a cap comprises m 7 G3 ⁇ OMepppCpC.
  • a cap comprises m 7 G 3 ⁇ OMe pppCpG. In some embodiments, a cap comprises m 7 G3 ⁇ OMepppCpU. In some embodiments, a cap comprises m 7 G3 ⁇ OMepppGpA. In some embodiments, a cap comprises m 7 G3 ⁇ OMepppGpC. In some embodiments, a cap comprises m 7 G 3 ⁇ OMe pppGpG. In some embodiments, a cap comprises m 7 G 3 ⁇ OMe pppGpU. In some embodiments, a cap comprises m 7 G 3 ⁇ OMe pppUpA.
  • a cap comprises m 7 G3 ⁇ OMepppUpC. In some embodiments, a cap comprises m 7 G3 ⁇ OMepppUpG. In some embodiments, a cap comprises m 7 G 3 ⁇ OMe pppUpU.
  • a cap in other embodiments, comprises a sequence selected from the following sequences: m 7 G3 ⁇ OMepppA2 ⁇ OMepA, m 7 G3 ⁇ OMepppA2 ⁇ OMepC, m 7 G 3 ⁇ OMe pppA 2 ⁇ OMe pG, m 7 G 3 ⁇ OMe pppA 2 ⁇ OMe pU, m 7 G 3 ⁇ OMe pppC 2 ⁇ OMe pA, m 7 G 3 ⁇ OMe pppC 2 ⁇ OMe pC, m 7 G 3 ⁇ OMe pppC 2 ⁇ OMe pG, m 7 G 3 ⁇ OMe pppC 2 ⁇ OMe pU, m 7 G3 ⁇ OMepppG2 ⁇ OMepA, m 7 G3 ⁇ OMepppG2 ⁇ OMepC, m 7 G3 ⁇ OMepC, m 7
  • a cap comprises m 7 G 3 ⁇ OMe pppA 2 ⁇ OMe pA. In some embodiments, a cap comprises m 7 G3 ⁇ OMepppA2 ⁇ OMepC. In some embodiments, a cap comprises m 7 G3 ⁇ OMepppA2 ⁇ OMepG. In some embodiments, a cap comprises m 7 G 3 ⁇ OMe pppA 2 ⁇ OMe pU. In some embodiments, a cap comprises m 7 G 3 ⁇ OMe pppC 2 ⁇ OMe pA. In some embodiments, a cap comprises m 7 G 3 ⁇ OMe pppC 2 ⁇ OMe pC.
  • a cap comprises m 7 G3 ⁇ OMepppC2 ⁇ OMepG. In some embodiments, a cap comprises m 7 G3 ⁇ OMepppC2 ⁇ OMepU. In some embodiments, a cap comprises m 7 G 3 ⁇ OMe pppG 2 ⁇ OMe pA. In some embodiments, a cap comprises m 7 G 3 ⁇ OMe pppG 2 ⁇ OMe pC. In some embodiments, a cap comprises m 7 G3 ⁇ OMepppG2 ⁇ OMepG.
  • a cap comprises Attorney Docket No.45817-0124WO1 / MTX959.20 m 7 G3 ⁇ OMepppG2 ⁇ OMepU. In some embodiments, a cap comprises m 7 G 3 ⁇ OMe pppU 2 ⁇ OMe pA. In some embodiments, a cap comprises m 7 G3 ⁇ OMepppU2 ⁇ OMepC. In some embodiments, a cap comprises m 7 G3 ⁇ OMepppU2 ⁇ OMepG. In some embodiments, a cap comprises m 7 G3 ⁇ OMepppU2 ⁇ OMepU.
  • a cap in still other embodiments, comprises a sequence selected from the following sequences: m 7 GpppG2 ⁇ OMe, m 7 GpppA2 ⁇ OMepA, m 7 GpppA2 ⁇ OMepC, m 7 GpppA 2 ⁇ OMe pG, m 7 GpppA 2 ⁇ OMe pU, m 7 GpppC 2 ⁇ OMe pA, m 7 GpppC 2 ⁇ OMe pC, m 7 GpppC 2 ⁇ OMe pG, m 7 GpppC 2 ⁇ OMe pU, m 7 GpppG 2 ⁇ OMe pA, m 7 GpppG 2 ⁇ OMe pC, m 7 GpppG2 ⁇ OMepG, m 7 GpppG2 ⁇ OMepU, m 7 GpppU2 ⁇ OMepA, m 7 GpppG2 ⁇ OMepG
  • a cap comprises m 7 GpppA 2 ⁇ OMe pA. In some embodiments, a cap comprises m 7 GpppA 2 ⁇ OMe pC. In some embodiments, a cap comprises m 7 GpppA2 ⁇ OMepG. In some embodiments, a cap comprises m 7 GpppA2 ⁇ OMepU. In some embodiments, a cap comprises m 7 GpppC2 ⁇ OMepA. In some embodiments, a cap comprises m 7 GpppC 2 ⁇ OMe pC. In some embodiments, a cap comprises m 7 GpppC 2 ⁇ OMe pG.
  • a trinucleotide cap comprises m 7 GpppC2 ⁇ OMepU. In some embodiments, a cap comprises m 7 GpppG2 ⁇ OMepA. In some embodiments, a cap comprises m 7 GpppG2 ⁇ OMepC. In some embodiments, a cap comprises m 7 GpppG 2 ⁇ OMe pG. In some embodiments, a cap comprises m 7 GpppG 2 ⁇ OMe pU. In some embodiments, a cap comprises m 7 GpppU 2 ⁇ OMe pA. In some embodiments, a cap comprises m 7 GpppU2 ⁇ OMepC.
  • a cap comprises m 7 GpppU2 ⁇ OMepG. In some embodiments, a cap comprises m 7 GpppU 2 ⁇ OMe pU. [00368] In some embodiments, a cap comprises m 7 Gpppm 6 A 2’Ome pG. In some embodiments, a cap comprises m 7 Gpppe 6 A2’OmepG. [00369] In some embodiments, a cap comprises GAG. In some embodiments, a cap comprises GCG. In some embodiments, a cap comprises GUG. In some embodiments, a cap comprises GGG.
  • a cap comprises any one of the following structures: or .
  • the cap comprises m7 GpppN 1 N 2 N 3 , where N 1 , N 2 , and N3 are optional (i.e., can be absent or one or more can be present) and are independently a natural, a modified, or an unnatural nucleoside base.
  • m7 G is further methylated, e.g., at the 3’ position.
  • the m7 G comprises an O-methyl at the 3’ position.
  • N1, N2, and N3 if present, optionally, are independently an adenine, a uracil, a guanidine, a thymine, or a cytosine.
  • one or more (or all) of N1, N2, and N3, if present, are methylated, e.g., at the 2’ position.
  • one or more (or all) of N1, N2, and N3, if present have an O-methyl at the 2’ position.
  • the cap comprises the following structure: unnatural nucleoside based; and R1, R2, R3, and R4 are independently OH or O- methyl.
  • R 3 is O-methyl and R 4 is OH.
  • R3 and R4 are O-methyl.
  • R4 is O-methyl.
  • R 1 is OH
  • R 2 is OH
  • R 3 is O-methyl
  • R 4 is OH.
  • R1 is OH
  • R2 is OH
  • R3 is O-methyl
  • R4 is O-methyl.
  • at least one of R 1 and R 2 is O-methyl
  • R 3 is O-methyl
  • R 4 is OH.
  • R1 and R2 is O-methyl
  • R3 is O-methyl
  • R4 is O-methyl
  • B 1 , B 3 , and B 3 are natural nucleoside bases.
  • at least one of B1, B2, and B3 is a modified or unnatural base.
  • at least one of B 1 , B 2 , and B 3 is N6-methyladenine.
  • B1 is adenine, cytosine, thymine, or uracil.
  • B1 is adenine
  • B 2 is uracil
  • B 3 is adenine.
  • R 1 and R 2 are OH, R3 and R4 are O-methyl, B1 is adenine, B2 is uracil, and B3 is adenine.
  • the cap comprises a sequence selected from the following sequences: GAAA, GACA, GAGA, GAUA, GCAA, GCCA, GCGA, GCUA, GGAA, GGCA, GGGA, GGUA, GUCA, and GUUA.
  • the cap comprises a sequence selected from the following sequences: GAAG, GACG, Attorney Docket No.45817-0124WO1 / MTX959.20 GAGG, GAUG, GCAG, GCCG, GCGG, GCUG, GGAG, GGCG, GGGG, GGUG, GUCG, GUGG, and GUUG.
  • the cap comprises a sequence selected from the following sequences: GAAU, GACU, GAGU, GAUU, GCAU, GCCU, GCGU, GCUU, GGAU, GGCU, GGGU, GGUU, GUAU, GUCU, GUGU, and GUUU.
  • the cap comprises a sequence selected from the following sequences: GAAC, GACC, GAGC, GAUC, GCAC, GCCC, GCGC, GCUC, GGAC, GGCC, GGGC, GGUC, GUAC, GUCC, GUGC, and GUUC.
  • a cap in some embodiments, comprises a sequence selected from the following sequences: m 7 G 3 ⁇ OMe pppApApN, m 7 G 3 ⁇ OMe pppApCpN, m 7 G 3 ⁇ OMe pppApGpN, m 7 G 3 ⁇ OMe pppApUpN, m 7 G 3 ⁇ OMe pppCpApN, m 7 G3 ⁇ OMepppCpCpN, m 7 G3 ⁇ OMepppCpGpN, m 7 G3 ⁇ OMepppCpUpN, m 7 G3 ⁇ OMepppGpApN, m 7 G3 ⁇ OMepppGpCpN, m 7 G3 ⁇ OMepppGpGpN, m 7 G3 ⁇ OMepppGpGpN, m 7 G3 ⁇ OMepppGpGpN, m 7 G
  • a cap in other embodiments, comprises a sequence selected from the following sequences: m 7 G3 ⁇ OMepppA2 ⁇ OMepApN, m 7 G3 ⁇ OMepppA2 ⁇ OMepCpN, m 7 G 3 ⁇ OMe pppA 2 ⁇ OMe pGpN, m 7 G 3 ⁇ OMe pppA 2 ⁇ OMe pUpN, m 7 G 3 ⁇ OMe pppC 2 ⁇ OMe pApN, m 7 G 3 ⁇ OMe pppC 2 ⁇ OMe pCpN, m 7 G 3 ⁇ OMe pppC 2 ⁇ OMe pGpN, m 7 G 3 ⁇ OMe pppC 2 ⁇ OMe pUpN, m 7 G3 ⁇ OMepppG2 ⁇ OMepApN, m 7 G3 ⁇ OMepppG2 ⁇ ⁇ OMep
  • a cap in still other embodiments, comprises a sequence selected from the following sequences: m 7 GpppA 2 ⁇ OMe pApN, m 7 GpppA 2 ⁇ OMe pCpN, m 7 GpppA2 ⁇ OMepGpN, m 7 GpppA2 ⁇ OMepUpN, m 7 GpppC2 ⁇ OMepApN, m 7 GpppC2 ⁇ OMepCpN, m 7 GpppC2 ⁇ OMepGpN, m 7 GpppC2 ⁇ OMepUpN, m 7 GpppG 2 ⁇ OMe pApN, m 7 GpppG 2 ⁇ OMe pCpN, m 7 GpppG 2 ⁇ OMe pG 2 , m 7 GpppG 2 ⁇ OMe pGpN, m 7 GpppG 2 ⁇ OMe pUpN, m 7 G
  • a cap in other embodiments, comprises a sequence selected from the following sequences: m 7 G3 ⁇ OMepppA2 ⁇ OMepA2 ⁇ OMepN, m 7 G3 ⁇ OMepppA2 ⁇ OMepC2 ⁇ OMepN, m 7 G3 ⁇ OMepppA2 ⁇ OMepG2 ⁇ OMepN, m 7 G3 ⁇ OMepppA2 ⁇ OMepU2 ⁇ OMepN, m 7 G 3 ⁇ OMe pppC 2 ⁇ OMe pA 2 ⁇ OMe pN, m 7 G 3 ⁇ OMe pppC 2 ⁇ OMe pC 2 ⁇ OMe pN, m 7 G 3 ⁇ OMe pppC 2 ⁇ OMe pG 2 ⁇ OMe pN, m 7 G 3 ⁇ OMe pppC 2 ⁇ OMe pG 2 ⁇ OMe
  • a cap in still other embodiments, comprises a sequence selected from the following sequences: m 7 GpppA2 ⁇ OMepA2 ⁇ OMepN, m 7 GpppA2 ⁇ OMepC2 ⁇ OMepN, m 7 GpppA2 ⁇ OMepG2 ⁇ OMepN, m 7 GpppA2 ⁇ OMepU2 ⁇ OMepN, m 7 GpppC2 ⁇ OMepA2 ⁇ OMepN, m 7 GpppC 2 ⁇ OMe pC 2 ⁇ OMe pN, m 7 GpppC 2 ⁇ OMe pG 2 ⁇ OMe pN, m 7 GpppC 2 ⁇ OMe pU 2 ⁇ OMe pN, m 7 GpppG 2 ⁇ OMe pA 2 ⁇ OMe pN, m 7 GpppG 2 ⁇ OMe pN, m 7
  • a cap comprises GGAG.
  • a cap comprises the following structure: (X). Attorney Docket No.45817-0124WO1 / MTX959.20 13.
  • Poly-A Tails [00381]
  • the polynucleotides of the present disclosure e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide
  • the polynucleotides of the present disclosure further comprise a poly-A tail.
  • terminal groups on the poly-A tail can be incorporated for stabilization.
  • a poly-A tail comprises des-3′ hydroxyl tails.
  • RNA processing a long chain of adenine nucleotides (poly-A tail) can be added to a polynucleotide such as an mRNA molecule in order to increase stability. Immediately after transcription, the 3′ end of the transcript can be cleaved to free a 3′ hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides to the RNA.
  • polyadenylation adds a poly-A tail that can be between, for example, approximately 80 to approximately 250 residues long, including approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 residues long.
  • the poly-A tail is 100 nucleotides in length (SEQ ID NO:195).
  • PolyA tails can also be added after the construct is exported from the nucleus.
  • terminal groups on the poly A tail can be incorporated for stabilization.
  • Polynucleotides of the present invention can include des-3′ hydroxyl tails.
  • the polynucleotides of the present invention can be designed to encode transcripts with alternative polyA tail structures including histone mRNA. According to Norbury, "Terminal uridylation has also been detected on human replication- dependent histone mRNAs. The turnover of these mRNAs is thought to be important for the prevention of potentially toxic histone accumulation following the completion or inhibition of chromosomal DNA replication.
  • mRNAs are distinguished by their lack of a 3 ⁇ poly(A) tail, the function of which is instead assumed by a stable Attorney Docket No.45817-0124WO1 / MTX959.20 stem–loop structure and its cognate stem–loop binding protein (SLBP); the latter carries out the same functions as those of PABP on polyadenylated mRNAs" (Norbury, "Cytoplasmic RNA: a case of the tail wagging the dog," Nature Reviews Molecular Cell Biology; AOP, published online 29 August 2013; doi:10.1038/nrm3645) the contents of which are incorporated herein by reference in its entirety.
  • SLBP stem–loop binding protein
  • the length of a poly-A tail when present, is greater than 30 nucleotides in length. In another embodiment, the poly-A tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides).
  • the poly-A tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600,
  • the polynucleotide or region thereof includes from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 2,500, from 1,500 to
  • the poly-A tail is designed relative to the length of the overall polynucleotide or the length of a particular region of the polynucleotide. This design can be based on the length of a coding region, the length of a particular feature or region or based on the length of the ultimate product expressed from the polynucleotides.
  • the poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the polynucleotide or feature thereof.
  • the poly-A tail can also be designed as a fraction of the polynucleotides to which it belongs.
  • the poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct, a construct region or the total length of the construct minus the poly-A tail.
  • engineered binding sites and conjugation of polynucleotides for Poly-A binding protein can enhance expression.
  • multiple distinct polynucleotides can be linked together via the PABP (Poly-A binding protein) through the 3′-end using modified nucleotides at the 3′-terminus of the poly-A tail.
  • the polynucleotides of the present invention are designed to include a polyA-G quartet region.
  • the G-quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA.
  • the G-quartet is incorporated at the end of the poly-A tail.
  • the resultant polynucleotide is assayed for stability, protein production and other parameters including half-life at various time points.
  • the polyA-G quartet results in protein production from an mRNA equivalent to at least 75% of that seen using a poly-A tail of 120 nucleotides alone (SEQ ID NO:196).
  • the polyA tail comprises an alternative nucleoside, e.g., inverted thymidine.
  • PolyA tails comprising an alternative nucleoside, e.g., inverted thymidine may be generated as described herein. For instance, mRNA constructs may be modified by ligation to stabilize the poly(A) tail.
  • Ligation may be performed using 0.5-1.5 mg/mL mRNA (5′ Cap1, 3′ A100), 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM TCEP, 1000 units/mL T4 RNA Ligase 1, 1 mM ATP, 20% w/v polyethylene glycol 8000, and 5:1 molar ratio of modifying oligo to mRNA.
  • Modifying oligo has a sequence of 5’-phosphate-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- (see below). Ligation reactions are mixed and incubated at room temperature ( ⁇ 22°C) for, e.g., 4 hours.
  • Stable tail mRNA are purified by, e.g., dT purification, reverse phase purification, hydroxyapatite purification, ultrafiltration into water, and sterile filtration.
  • the resulting stable tail-containing mRNAs contain the following structure at the 3’end, Attorney Docket No.45817-0124WO1 / MTX959.20 starting with the polyA region: A100-UCUAGAAAAAAAAAAAAAAAAAA- inverted deoxythymidine (SEQ ID NO:211).
  • the polyA tail comprises A100-UCUAG-A20-inverted deoxy-thymidine (SEQ ID NO:211). In some instances, the polyA tail consists of A100-UCUAG-A20-inverted deoxy-thymidine (SEQ ID NO:211). 14.
  • the invention also includes a polynucleotide that comprises both a start codon region and the polynucleotide described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide).
  • the polynucleotides of the present invention can have regions that are analogous to or function like a start codon region.
  • the translation of a polynucleotide can initiate on a codon that is not the start codon AUG.
  • Translation of the polynucleotide can initiate on an alternative start codon such as, but not limited to, ACG, AGG, AAG, CTG/CUG, GTG/GUG, ATA/AUA, ATT/AUU, TTG/UUG (see Touriol et al. Biology of the Cell 95 (2003) 169-178 and Matsuda and Mauro PLoS ONE, 2010 5:11; the contents of each of which are herein incorporated by reference in its entirety).
  • Attorney Docket No.45817-0124WO1 / MTX959.20 [00397]
  • the translation of a polynucleotide begins on the alternative start codon ACG.
  • polynucleotide translation begins on the alternative start codon CTG or CUG.
  • the translation of a polynucleotide begins on the alternative start codon GTG or GUG.
  • Nucleotides flanking a codon that initiates translation such as, but not limited to, a start codon or an alternative start codon, are known to affect the translation efficiency, the length and/or the structure of the polynucleotide. (See, e.g., Matsuda and Mauro PLoS ONE, 20105:11; the contents of which are herein incorporated by reference in its entirety).
  • Masking any of the nucleotides flanking a codon that initiates translation can be used to alter the position of translation initiation, translation efficiency, length and/or structure of a polynucleotide.
  • a masking agent can be used near the start codon or alternative start codon in order to mask or hide the codon to reduce the probability of translation initiation at the masked start codon or alternative start codon.
  • Non-limiting examples of masking agents include antisense locked nucleic acids (LNA) polynucleotides and exon-junction complexes (EJCs) (See, e.g., Matsuda and Mauro describing masking agents LNA polynucleotides and EJCs (PLoS ONE, 20105:11); the contents of which are herein incorporated by reference in its entirety).
  • LNA antisense locked nucleic acids
  • EJCs exon-junction complexes
  • a masking agent can be used to mask a start codon of a polynucleotide in order to increase the likelihood that translation will initiate on an alternative start codon.
  • a masking agent can be used to mask a first start codon or alternative start codon in order to increase the chance that translation will initiate on a start codon or alternative start codon downstream to the masked start codon or alternative start codon.
  • a start codon or alternative start codon can be located within a perfect complement for a miRNA binding site.
  • the perfect complement of a miRNA binding site can help control the translation, length and/or structure of the polynucleotide similar to a masking agent.
  • the start codon or alternative start codon can be located in the middle of a perfect complement for a miRNA binding site.
  • the start codon or alternative start codon can be located after the first nucleotide, second nucleotide, third nucleotide, fourth Attorney Docket No.45817-0124WO1 / MTX959.20 nucleotide, fifth nucleotide, sixth nucleotide, seventh nucleotide, eighth nucleotide, ninth nucleotide, tenth nucleotide, eleventh nucleotide, twelfth nucleotide, thirteenth nucleotide, fourteenth nucleotide, fifteenth nucleotide, sixteenth nucleotide, seventeenth nucleotide, eighteenth nucleotide, nineteenth nucleotide, twentieth nucleotide or twenty-first nucleotide.
  • the start codon of a polynucleotide can be removed from the polynucleotide sequence in order to have the translation of the polynucleotide begin on a codon that is not the start codon. Translation of the polynucleotide can begin on the codon following the removed start codon or on a downstream start codon or an alternative start codon.
  • the start codon ATG or AUG is removed as the first 3 nucleotides of the polynucleotide sequence in order to have translation initiate on a downstream start codon or alternative start codon.
  • the polynucleotide sequence where the start codon was removed can further comprise at least one masking agent for the downstream start codon and/or alternative start codons in order to control or attempt to control the initiation of translation, the length of the polynucleotide and/or the structure of the polynucleotide.
  • Stop Codon Region [00403] The invention also includes a polynucleotide that comprises both a stop codon region and the polynucleotide described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide).
  • the polynucleotides of the present invention can include at least two stop codons before the 3′ untranslated region (UTR).
  • the stop codon can be selected from TGA, TAA and TAG in the case of DNA, or from UGA, UAA and UAG in the case of RNA.
  • the polynucleotides of the present invention include the stop codon TGA in the case or DNA, or the stop codon UGA in the case of RNA, and one additional stop codon.
  • the addition stop codon can be TAA or UAA.
  • the polynucleotides of the present invention include three consecutive stop codons, four stop codons, or more.
  • any of the polynucleotides disclosed herein can comprise one, two, three, or all of the following elements: (a) a 5’-UTR, e.g., as described herein; (b) a coding region comprising a stop element (e.g., as described herein); (c) a 3’-UTR (e.g., as described herein) and; optionally (d) a 3’ stabilizing region, e.g., as described herein. Also disclosed herein are LNP compositions comprising the same.
  • a polynucleotide of the disclosure comprises (a) a 5’ UTR described in Table 4 or a variant or fragment thereof and (b) a coding region comprising a stop element provided herein.
  • the polynucleotide further comprises a cap structure, e.g., as described herein, or a poly A tail, e.g., as described herein.
  • the polynucleotide further comprises a 3’ stabilizing region, e.g., as described herein.
  • a polynucleotide of the disclosure comprises (a) a 5’ UTR described in Table 4 or a variant or fragment thereof and (c) a 3’ UTR described in Table 5 or Table 7 or a variant or fragment thereof.
  • the polynucleotide further comprises a cap structure, e.g., as described herein, or a poly A tail, e.g., as described herein.
  • the polynucleotide further comprises a 3’ stabilizing region, e.g., as described herein.
  • a polynucleotide of the disclosure comprises (c) a 3’ UTR described in Table 5 or Table 7 or a variant or fragment thereof and (b) a coding region comprising a stop element provided herein.
  • the polynucleotide comprises a sequence provided in Table 7.
  • the polynucleotide further comprises a cap structure, e.g., as described herein, or a poly A tail, e.g., as described herein.
  • the polynucleotide further comprises a 3’ stabilizing region, e.g., as described herein.
  • a polynucleotide of the disclosure comprises (a) a 5’ UTR described in Table 4 or a variant or fragment thereof; (b) a coding region comprising a stop element provided herein; and (c) a 3’ UTR described in Table 5 or Table 7 or a variant or fragment thereof.
  • the polynucleotide further comprises a cap structure, e.g., as described herein, or a poly A tail, e.g., as Attorney Docket No.45817-0124WO1 / MTX959.20 described herein.
  • the polynucleotide further comprises a 3’ stabilizing region, e.g., as described herein.
  • Table 7 Exemplary 3’ UTR and stop element sequences SEQ ID Sequence NO information Sequence U C U U C U U C U U C U U C U U C U U C U U C U U C U U C U U C U U G C C C Attorney Docket No.45817-0124WO1 / MTX959.20 CCCUCCAUAAAGUAGGAAACACUACAGUGGUC UUUGAAUAAAGUCUGAGUGGGCGGC ’ C C C U G C C C U G C U C C C C U G U C U C U 17.
  • An Identification and Ratio Determination (IDR) sequence is a sequence of a biological molecule (e.g., nucleic acid or protein) that, when combined with the sequence of a target biological molecule, serves to identify the target biological molecule.
  • an IDR sequence is a heterologous sequence that is incorporated within or appended to a sequence of a target biological molecule and can be used as a reference to identify the target molecule.
  • a nucleic acid comprises (i) a target sequence of interest (e.g., a coding sequence Attorney Docket No.45817-0124WO1 / MTX959.20 encoding a therapeutic and/or antigenic peptide or protein); and (ii) a unique IDR sequence.
  • a target sequence of interest e.g., a coding sequence Attorney Docket No.45817-0124WO1 / MTX959.20 encoding a therapeutic and/or antigenic peptide or protein
  • a unique IDR sequence e.g., an RNA species (e.g., RNA having a given coding sequence) may comprise an IDR sequence that differs from the IDR sequence of other RNA species (e.g., RNA(s) having different coding sequence(s)).
  • Each IDR sequence thus identifies a particular RNA species, and so the abundance of IDR sequences may be measured to determine the abundance of each RNA species in a composition.
  • Use of distinct IDR sequences to identify RNA species allows for analysis of multivalent RNA compositions (e.g., containing multiple RNA species) containing RNA species with similar coding sequences and/or lengths, which could otherwise be difficult to distinguish using PCR- or chromatography-based analysis of full-length RNAs.
  • Each RNA species in a multivalent RNA composition may comprise an IDR sequence that is not a sequence isomer of an IDR sequence of another RNA species in a multivalent RNA composition (e.g., the IDR sequence does not have the same number of adenosine nucleotides, the same number of cytosine nucleotides, the same number of guanine nucleotides, and the same number of uracil nucleotides, as another IDR sequence in the composition, even if those sequences have different sequences).
  • Each RNA species in a multivalent RNA composition may comprise an IDR sequence having a mass that differs from the mass of IDR sequences of each other RNA species in a multivalent RNA composition.
  • the mass of each IDR sequence may differ from the mass of other IDR sequences by at least 9 Da, at least 25 Da, at least 25 Da, or at least 50 Da.
  • RNA fragments comprising different IDR sequences may be distinguished using mass-based analysis methods (e.g., mass spectrometry), which do not require reverse transcription, amplification, or sequencing of RNAs.
  • mass-based analysis methods e.g., mass spectrometry
  • Each RNA species in an RNA composition may comprises an IDR sequence with a different length.
  • each IDR sequence may have a length independently selected from 0 to 25 nucleotides.
  • IDR sequences may be chosen such that no IDR sequence comprises a start codon, ‘AUG’. Lack of a start codon in an IDR sequence prevents undesired translation of nucleotide sequences within and/or downstream from the IDR sequence.
  • IDR sequences may be chosen such that no IDR sequence comprises a recognition site for a restriction enzyme.
  • no IDR sequence comprises a recognition site for XbaI, ‘UCUAG’.
  • Lack of a recognition site for a restriction enzyme e.g., XbaI recognition site ‘UCUAG’) allows the restriction enzyme to be used in generating and modifying a DNA template for in vitro transcription, without affecting the IDR sequence or sequence of the transcribed RNA. 18.
  • a polynucleotide of the present disclosure comprises from 5′ to 3′ end: (i) a 5′ cap; (ii) a 5′ UTR; (iii) an ORF encoding a polypeptide comprising any one of SEQ ID NOs:320- 373; (iv) at least one stop codon; (v) a 3′ UTR; and (vi) a poly-A tail.
  • a polynucleotide of the present disclosure for example a polynucleotide comprising an mRNA nucleotide sequence encoding a Snu13 polypeptide, comprises from 5′ to 3′ end: (i) a 5′ cap such as provided above; (ii) a 5′ UTR, such as provided above; (iii) an ORF encoding a polypeptide comprising a Snu13 polypeptide (e.g., any one of SEQ ID NOs:320-373), wherein the ORF comprises a sequence that has at least 65%, at least 70%, at least 75%, at least 80%, at least 85, at least 90%, at least Attorney Docket No.45817-0124WO1 / MTX959.20 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of any one of SEQ ID NOs: 300-317; (iv) at least one stop codon;
  • a polynucleotide of the present disclosure for example a polynucleotide comprising an mRNA nucleotide sequence encoding a Snu13 polypeptide, comprises from 5′ to 3′ end: (i) a 5′ cap such as provided above; (ii) a 5′ UTR such as provided above; (iii) an ORF comprising the sequence of any one of SEQ ID NOs:300-317; (iv) at least one stop codon; (v) a 3′ UTR such as provided above; and (vi) a poly-A tail provided above.
  • a polynucleotide of the present disclosure for example a polynucleotide comprising an mRNA nucleotide sequence encoding a Snu13 polypeptide, comprises from 5′ to 3′ end: (i) a 5′ cap such as provided above; (ii) a 5′ UTR comprising the sequence set forth in SEQ ID NO:50; (iii) an ORF encoding a Snu13 polypeptide (e.g., any one of SEQ ID NOs:320-373), wherein the ORF has at least 65%, at least 70%, at least 75%, at least 80%, at least 85, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of any one of SEQ ID NOs: 300-317; (iv) at least one stop codon; (v) a 3′ UTR comprising the sequence set forth in SEQ ID NO:108; and (vi
  • a polynucleotide of the present disclosure for example a polynucleotide comprising an mRNA nucleotide sequence encoding a Snu13 polypeptide, comprises from 5′ to 3′ end: (i) a 5′ cap such as provided above; (ii) a 5′ UTR comprising the sequence set forth in SEQ ID NO:50; Attorney Docket No.45817-0124WO1 / MTX959.20 (iii) an ORF encoding a Snu13 polypeptide (e.g., any one of SEQ ID NOs:320-373), wherein the ORF has at least 65%, at least 70%, at least 75%, at least 80%, at least 85, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of any one of SEQ ID NOs: 300-317; (iv) at least one stop codon; (v)
  • the polynucleotide further comprises a miRNA binding site, e.g., a miRNA binding site that binds to miRNA-142.
  • the 3′ UTR comprises the miRNA binding site.
  • a polynucleotide of the present disclosure comprises a nucleotide sequence encoding a polypeptide sequence at least 65%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , at least 97%, at least 98%, at least 99%, or 100% identical to the protein sequence of a variant Snu13 polypeptide described herein (e.g., any one of SEQ ID NOs:320-373, wherein the encoded polypeptide retains the substitutions present any one of SEQ ID NOs:320- 373, respectively, relative to wild type Snu13).
  • a variant Snu13 polypeptide described herein e.g., any one of SEQ ID NOs:320-373, where
  • a polynucleotide of the present disclosure for example a polynucleotide comprising an mRNA nucleotide sequence encoding a polypeptide, comprises (1) a 5′ cap such as provided above, for example, m7Gp- ppGm-A, (2) a 5′ UTR, (3) a nucleotide sequence ORF comprising the sequence of any one of SEQ ID NOs: 300-317, (3) a stop codon, (4) a 3′UTR, and (5) a poly-A tail provided above, for example, a poly-A tail of SEQ ID NO:195 or A100-UCUAG- A20-inverted deoxy-thymidine (SEQ ID NO:211).
  • SEQ ID NO: 374 consists from 5′ to 3′ end: 5′ UTR of SEQ ID NO: 50, Snu13 nucleotide ORF of SEQ ID NO: 300, and 3′ UTR of SEQ ID NO: 108.
  • SEQ ID NO: 375 consists from 5′ to 3′ end: 5′ UTR of SEQ ID NO: 50, Snu13 nucleotide ORF of SEQ ID NO: 301, and 3′ UTR of SEQ ID NO: 108.
  • SEQ ID NO: 376 consists from 5′ to 3′ end: 5′ UTR of SEQ ID NO: 50, Snu13 nucleotide ORF of SEQ ID NO: 302, and 3′ UTR of SEQ ID NO: 108.
  • SEQ ID NO: 377 consists from 5′ to 3′ end: 5′ UTR of SEQ ID NO: 50, Snu13 nucleotide ORF of SEQ ID NO: 303, and 3′ UTR of SEQ ID NO: 108.
  • SEQ ID NO: 378 consists from 5′ to 3′ end: 5′ UTR of SEQ ID NO: 50, Snu13 nucleotide ORF of SEQ ID NO: 304, and 3′ UTR of SEQ ID NO: 108.
  • SEQ ID NO: 379 consists from 5′ to 3′ end: 5′ UTR of SEQ ID NO: 50, Snu13 nucleotide ORF of SEQ ID NO: 305, and 3′ UTR of SEQ ID NO: 108.
  • SEQ ID NO: 380 consists from 5′ to 3′ end: 5′ UTR of SEQ ID NO: 50, Snu13 nucleotide ORF of SEQ ID NO: 306, and 3′ UTR of SEQ ID NO: 108.
  • SEQ ID NO: 381 consists from 5′ to 3′ end: 5′ UTR of SEQ ID NO: 50, Snu13 nucleotide ORF of SEQ ID NO: 307, and 3′ UTR of SEQ ID NO: 108.
  • SEQ ID NO: 382 consists from 5′ to 3′ end: 5′ UTR of SEQ ID NO: 50, Snu13 nucleotide ORF of SEQ ID NO: 308, and 3′ UTR of SEQ ID NO: 108.
  • SEQ ID NO: 383 consists from 5′ to 3′ end: 5′ UTR of SEQ ID NO: 50, Snu13 nucleotide ORF of SEQ ID NO: 309, and 3′ UTR of SEQ ID NO: 108.
  • SEQ ID NO: 384 consists from 5′ to 3′ end: 5′ UTR of SEQ ID NO: 50, Snu13 nucleotide ORF of SEQ ID NO: 310, and 3′ UTR of SEQ ID NO: 108.
  • SEQ ID NO: 385 consists from 5′ to 3′ end: 5′ UTR of SEQ ID NO: 50, Snu13 nucleotide ORF of SEQ ID NO: 311, and 3′ UTR of SEQ ID NO: 108.
  • SEQ ID NO: 386 consists from 5′ to 3′ end: 5′ UTR of SEQ ID NO: 50, Snu13 nucleotide ORF of SEQ ID NO: 312, and 3′ UTR of SEQ ID NO: 108.
  • SEQ ID NO: 387 consists from 5′ to 3′ end: 5′ UTR of SEQ ID NO: 50, Snu13 nucleotide ORF of SEQ ID NO: 313, and 3′ UTR of SEQ ID NO: 108.
  • SEQ ID NO: 388 consists from 5′ to 3′ end: 5′ UTR of SEQ ID NO: 50, Snu13 nucleotide ORF of SEQ ID NO: 314, and 3′ UTR of SEQ ID NO: 108.
  • SEQ ID NO: 389 consists from 5′ to 3′ end: 5′ UTR of SEQ ID NO: 50, Snu13 nucleotide ORF of SEQ ID NO: 315, and 3′ UTR of SEQ ID NO: 108.
  • SEQ ID NO: 390 consists from 5′ to 3′ end: 5′ UTR of SEQ ID NO: 50, Snu13 nucleotide ORF of SEQ ID NO: 316, and 3′ UTR of SEQ ID NO: 108.
  • SEQ ID NO: 391 consists from 5′ to 3′ end: 5′ UTR of SEQ ID NO: 50, Snu13 nucleotide ORF of SEQ ID NO: 317, and 3′ UTR of SEQ ID NO: 108.
  • Additional exemplary Snu13 nucleotide constructs include SEQ ID NOs:374-391, except wherein the construct has 3′ UTR of SEQ ID NO: 139 instead of SEQ ID NO:108.
  • all uracils therein are replaced by N1 methylpseudouracil.
  • all uracils therein are replaced by 5- methoxyuracil.
  • a polynucleotide of the present disclosure for example a polynucleotide comprising an mRNA nucleotide sequence encoding a Snu13 polypeptide, comprises (1) a 5′ cap such as provided above, for example, m 7 Gp-ppGm-A, (2) a nucleotide sequence of any one of SEQ ID NOs: 374-391, and (3) a poly-A tail provided above, for example, a poly A tail of ⁇ 100 residues, e.g., SEQ ID NO:195 or A100-UCUAG-A20-inverted deoxy-thymidine (SEQ ID NO:211).
  • a polynucleotide of the invention e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide
  • a polynucleotide e.g., a RNA, e.g., an mRNA
  • IVT in vitro transcription
  • a polynucleotide e.g., a RNA, e.g., an mRNA
  • a polynucleotide can be constructed by chemical synthesis using an oligonucleotide synthesizer.
  • a polynucleotide e.g., a RNA, e.g., an mRNA
  • encoding a Snu13 polypeptide is made by using a host cell.
  • a polynucleotide e.g., a RNA, e.g., an mRNA
  • Attorney Docket No.45817-0124WO1 / MTX959.20 encoding a Snu13 polypeptide is made by one or more combination of the IVT, chemical synthesis, host cell expression, or any other methods known in the art.
  • Naturally occurring nucleosides, non-naturally occurring nucleosides, or combinations thereof, can totally or partially naturally replace occurring nucleosides present in the candidate nucleotide sequence and can be incorporated into a sequence- optimized nucleotide sequence (e.g., a RNA, e.g., an mRNA) encoding a Snu13 polypeptide.
  • the resultant polynucleotides, e.g., mRNAs can then be examined for their ability to produce protein and/or produce a therapeutic outcome.
  • a. In Vitro Transcription / Enzymatic Synthesis [00435]
  • the present disclosure also provides methods for making a polynucleotide disclosed herein or a complement thereof.
  • a polynucleotide (e.g., an mRNA) disclosed herein can be constructed using in vitro transcription.
  • a polynucleotide (e.g., an mRNA) disclosed herein can be constructed by chemical synthesis using an oligonucleotide synthesizer.
  • a polynucleotide (e.g., an mRNA) disclosed herein is made by using a host cell.
  • a polynucleotide (e.g., an mRNA) disclosed herein is made by one or more combination of the IVT, chemical synthesis, host cell expression, or any other methods known in the art.
  • Naturally occurring nucleosides, non-naturally occurring nucleosides, or combinations thereof, can totally or partially naturally replace occurring nucleosides present in the candidate nucleotide sequence and can be incorporated into a sequence- optimized nucleotide sequence (e.g., an mRNA) encoding a Snu13 polypeptide.
  • a sequence- optimized nucleotide sequence e.g., an mRNA
  • the resultant mRNAs can then be examined for their ability to produce Snu13 and/or produce a therapeutic outcome.
  • RNA transcript e.g., mRNA transcript
  • a RNA polymerase e.g., a T7 RNA polymerase or a T7 RNA polymerase variant
  • the present disclosure provides methods of performing an IVT (in vitro transcription) reaction, comprising contacting a DNA template with the Attorney Docket No.45817-0124WO1 / MTX959.20 RNA polymerase (e.g., a T7 RNA polymerase, such as a T7 RNA polymerase variant) in the presence of nucleoside triphosphates and buffer under conditions that result in the production of RNA transcripts.
  • RNA polymerase e.g., a T7 RNA polymerase, such as a T7 RNA polymerase variant
  • capping methods e.g., co- transcriptional capping methods or other methods known in the art.
  • a capping method comprises reacting a polynucleotide template with a T7 RNA polymerase variant, nucleoside triphosphates, and a cap analog under in vitro transcription reaction conditions to produce RNA transcript.
  • IVT conditions typically require a purified linear DNA template containing a promoter, nucleoside triphosphates, a buffer system that includes dithiothreitol (DTT) and magnesium ions, and a RNA polymerase. The exact conditions used in the transcription reaction depend on the amount of RNA needed for a specific application.
  • Typical IVT reactions are performed by incubating a DNA template with a RNA polymerase and nucleoside triphosphates, including GTP, ATP, CTP, and UTP (or nucleotide analogs) in a transcription buffer.
  • a RNA transcript having a 5 ⁇ terminal guanosine triphosphate is produced from this reaction.
  • a deoxyribonucleic acid (DNA) is simply a nucleic acid template for RNA polymerase.
  • a DNA template may include a polynucleotide encoding a Snu13 polypeptide.
  • a DNA template in some embodiments, includes a RNA polymerase promoter (e.g., a T7 RNA polymerase promoter) located 5' from and operably linked to polynucleotide encoding a Snu13 polypeptide.
  • a DNA template may also include a nucleotide sequence encoding a polyadenylation (polyA) tail located at the 3' end of the gene of interest.
  • Polypeptides of interest include, but are not limited to, biologics, antibodies, antigens (vaccines), and therapeutic proteins.
  • the term “protein” encompasses peptides.
  • a RNA transcript in some embodiments, is the product of an IVT reaction and, as will be understood by one of ordinary skill in the art, the DNA template for making an RNA molecule is known based on base complementarity.
  • a RNA transcript in some embodiments, is a messenger RNA (mRNA) that includes a nucleotide sequence encoding a polypeptide of interest linked to a polyA tail.
  • mRNA messenger RNA
  • the mRNA is modified mRNA (mmRNA), which includes at least one modified nucleotide.
  • a nucleotide includes a nitrogenous base, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group.
  • Nucleotides include nucleoside monophosphates, nucleoside diphosphates, and nucleoside triphosphates.
  • a nucleoside monophosphate (NMP) includes a nucleobase linked to a ribose and a single phosphate;
  • a nucleoside triphosphate (NTP) includes a nucleobase linked to a ribose and three phosphates.
  • Nucleotide analogs are compounds that have the general structure of a nucleotide or are structurally similar to a nucleotide. Nucleotide analogs, for example, include an analog of the nucleobase, an analog of the sugar and/or an analog of the phosphate group(s) of a nucleotide. [00446] A nucleoside includes a nitrogenous base and a 5-carbon sugar. Thus, a nucleoside plus a phosphate group yields a nucleotide. Nucleoside analogs are compounds that have the general structure of a nucleoside or are structurally similar to a nucleoside.
  • Nucleoside analogs include an analog of the nucleobase and/or an analog of the sugar of a nucleoside.
  • nucleotide includes naturally- occurring nucleotides, synthetic nucleotides and modified nucleotides, unless indicated otherwise.
  • examples of naturally-occurring nucleotides used for the production of RNA, e.g., in an IVT reaction, as provided herein include adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), uridine triphosphate (UTP), and 5-methyluridine triphosphate (m 5 UTP).
  • ATP adenosine triphosphate
  • GTP guanosine triphosphate
  • CTP cytidine triphosphate
  • UTP uridine triphosphate
  • m 5 UTP 5-methyluridine triphosphate
  • adenosine diphosphate ADP
  • GDP guanosine diphosphate
  • CDP cytidine diphosphate
  • UDP uridine diphosphate
  • nucleotide analogs include, but are not limited to, antiviral nucleotide analogs, phosphate analogs (soluble or immobilized, hydrolyzable or non- hydrolyzable), dinucleotide, trinucleotide, tetranucleotide, e.g., a cap analog, or a precursor/substrate for enzymatic capping (vaccinia or ligase), a nucleotide labeled with a functional group to facilitate ligation/conjugation of cap or 5 ⁇ moiety (IRES), a nucleotide labeled with a 5 ⁇ PO4 to facilitate ligation of cap or 5 ⁇ moiety, or a nucleotide label
  • antiviral nucleotide/nucleoside analogs include, but are not limited, to Ganciclovir, Entecavir, Telbivudine, Vidarabine and Cidofovir.
  • Modified nucleotides may include modified nucleobases.
  • RNA transcript e.g., mRNA transcript
  • a modified nucleobase selected from pseudouridine ( ⁇ ), 1-methylpseudouridine (m1 ⁇ ), 1-ethylpseudouridine, 2-thiouridine, 4’-thiouridine, 2-thio-1-methyl-1-deaza- pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine , 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2- thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5- methoxyuridine (mo5U) and 2’-O-methyl uridine.
  • pseudouridine ⁇
  • a RNA transcript (e.g., mRNA transcript) includes a combination of at least two (e.g., 2, 3, 4 or more) of the foregoing modified nucleobases.
  • the nucleoside triphosphates (NTPs) as provided herein may comprise unmodified or modified ATP, modified or unmodified UTP, modified or unmodified GTP, and/or modified or unmodified CTP.
  • NTPs of an IVT reaction comprise unmodified ATP.
  • NTPs of an IVT reaction comprise modified ATP.
  • NTPs of an IVT reaction comprise unmodified UTP.
  • NTPs of an IVT reaction comprise modified UTP.
  • NTPs of an IVT reaction comprise unmodified GTP. In some embodiments, NTPs of an IVT reaction comprise modified GTP. In some embodiments, NTPs of an IVT reaction comprise unmodified CTP. In some embodiments, NTPs of an IVT reaction comprise modified CTP. [00451]
  • concentration of nucleoside triphosphates and cap analog present in an IVT reaction may vary. In some embodiments, NTPs and cap analog are present in the reaction at equimolar concentrations. In some embodiments, the molar ratio of cap analog (e.g., trinucleotide cap) to nucleoside triphosphates in the reaction is greater than 1:1.
  • the molar ratio of cap analog to nucleoside triphosphates in the reaction may be 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 50:1, or 100:1.
  • the molar ratio of cap analog (e.g., trinucleotide cap) to nucleoside triphosphates in the reaction is less than 1:1.
  • the molar ratio Attorney Docket No.45817-0124WO1 / MTX959.20 of cap analog (e.g., trinucleotide cap) to nucleoside triphosphates in the reaction may be 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:50, or 1:100.
  • the composition of NTPs in an IVT reaction may also vary.
  • ATP may be used in excess of GTP, CTP and UTP.
  • an IVT reaction may include 7.5 millimolar GTP, 7.5 millimolar CTP, 7.5 millimolar UTP, and 3.75 millimolar ATP.
  • the same IVT reaction may include 3.75 millimolar cap analog (e.g., trinucleotide cap).
  • the molar ratio of G:C:U:A:cap is 1:1:1:0.5:0.5. In some embodiments, the molar ratio of G:C:U:A:cap is 1:1:0.5:1:0.5. In some embodiments, the molar ratio of G:C:U:A:cap is 1:0.5:1:1:0.5. In some embodiments, the molar ratio of G:C:U:A:cap is 0.5:1:1:1:0.5.
  • a RNA transcript (e.g., mRNA transcript) includes a modified nucleobase selected from pseudouridine ( ⁇ ), 1-methylpseudouridine (m 1 ⁇ ), 5-methoxyuridine (mo 5 U), 5-methylcytidine (m 5 C), ⁇ -thio-guanosine and ⁇ -
  • a RNA transcript (e.g., mRNA transcript) includes a combination of at least two (e.g., 2, 3, 4 or more) of the foregoing modified nucleobases.
  • a RNA transcript (e.g., mRNA transcript) includes pseudouridine ( ⁇ ).
  • a RNA transcript (e.g., mRNA transcript) includes 1-methylpseudouridine (m 1 ⁇ ). In some embodiments, a RNA transcript (e.g., mRNA transcript) includes 5-methoxyuridine (mo 5 U). In some embodiments, a RNA transcript (e.g., mRNA transcript) includes 5-methylcytidine (m 5 C). In some embodiments, a RNA transcript (e.g., mRNA transcript) includes ⁇ -thio-guanosine. In some embodiments, a RNA transcript (e.g., mRNA transcript) includes ⁇ -thio- adenosine.
  • the polynucleotide e.g., RNA polynucleotide, such as mRNA polynucleotide
  • RNA polynucleotide is uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification.
  • a polynucleotide can be uniformly modified with 1-methylpseudouridine (m 1 ⁇ ), meaning that all uridine residues in the mRNA sequence are replaced methylpseudouridine (m 1 ⁇ ).
  • m 1 ⁇ 1-methylpseudouridine
  • a polynucleotide can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as any of those set forth above.
  • the Attorney Docket No.45817-0124WO1 / MTX959.20 polynucleotide may not be uniformly modified (e.g., partially modified, part of the sequence is modified).
  • RNA polynucleotide such as mRNA polynucleotide
  • the buffer system contains tris.
  • the concentration of tris used in an IVT reaction may be at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM or at least 110 mM phosphate.
  • the concentration of phosphate is 20-60 mM or 10-100 mM.
  • the buffer system contains dithiothreitol (DTT).
  • DTT dithiothreitol
  • the concentration of DTT used in an IVT reaction for example, may be at least 1 mM, at least 5 mM, or at least 50 mM.
  • the concentration of DTT used in an IVT reaction is 1-50 mM or 5-50 mM. In some embodiments, the concentration of DTT used in an IVT reaction is 5 mM.
  • the buffer system contains magnesium.
  • the molar ratio of NTP to magnesium ions (Mg 2+ ; e.g., MgCl 2 ) present in an IVT reaction is 1:1 to 1:5. For example, the molar ratio of NTP to magnesium ions may be 1:1, 1:2, 1:3, 1:4 or 1:5.
  • the molar ratio of NTP plus cap analog (e.g., trinucleotide cap, such as GAG) to magnesium ions (Mg 2+ ; e.g., MgCl 2 ) present in an IVT reaction is 1:1 to 1:5.
  • the molar ratio of NTP+trinucleotide cap (e.g., GAG) to magnesium ions may be 1:1, 1:2, 1:3, 1:4 or 1:5.
  • the buffer system contains Tris-HCl, spermidine (e.g., at a concentration of 1-30 mM), TRITON ® X-100 (polyethylene glycol p- (1,1,3,3-tetramethylbutyl)-phenyl ether) and/or polyethylene glycol (PEG).
  • NTPs nucleoside triphosphates
  • the addition of nucleoside triphosphates (NTPs) to the 3 ⁇ end of a growing RNA strand is catalyzed by a polymerase, such as T7 RNA polymerase, for example, any one or more of the T7 RNA polymerase variants (e.g., G47A) of the present disclosure.
  • the RNA polymerase (e.g., T7 RNA polymerase variant) is present in a reaction (e.g., an IVT reaction) at a concentration of 0.01 mg/ml to 1 mg/ml.
  • a reaction e.g., an IVT reaction
  • the RNA polymerase may be present in a reaction at a concentration of 0.01 mg/mL, 0.05 mg/ml, 0.1 mg/ml, 0.5 mg/ml or 1.0 mg/ml.
  • Attorney Docket No.45817-0124WO1 / MTX959.20 [00462]
  • the polynucleotide of the present disclosure is an IVT polynucleotide.
  • the basic components of an mRNA molecule include at least a coding region, a 5′UTR, a 3′UTR, a 5′ cap and a poly-A tail.
  • the IVT polynucleotides of the present disclosure can function as mRNA but are distinguished from wild-type mRNA in their functional and/or structural design features which serve, e.g., to overcome existing problems of effective polypeptide production using nucleic-acid based therapeutics.
  • the primary construct of an IVT polynucleotide comprises a first region of linked nucleotides that is flanked by a first flanking region and a second flaking region.
  • This first region can include, but is not limited to, the encoded Snu13 polypeptide.
  • the first flanking region can include a sequence of linked nucleosides which function as a 5’ untranslated region (UTR) such as the 5’ UTR of SEQ ID NO:58.
  • the IVT encoding a Snu13 polypeptide can comprise at its 5 terminus a signal sequence region encoding one or more signal sequences.
  • the flanking region can comprise a region of linked nucleotides comprising one or more complete or incomplete 5′ UTRs sequences.
  • the flanking region can also comprise a 5′ terminal cap.
  • the second flanking region can comprise a region of linked nucleotides comprising one or more complete or incomplete 3′ UTRs which can encode the native 3’ UTR of a Snu13 polypeptide, or a non-native 3’ UTR such as, but not limited to, a heterologous 3’ UTR or a synthetic 3’ UTR.
  • the flanking region can also comprise a 3′ tailing sequence.
  • the 3’ tailing sequence can be, but is not limited to, a polyA tail, a polyA-G quartet and/or a stem loop sequence.
  • RNA oligomer containing a codon- Attorney Docket No.45817-0124WO1 / MTX959.20 optimized nucleotide sequence coding for the particular isolated polypeptide can be synthesized.
  • several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated.
  • the individual oligonucleotides typically contain 5′ or 3′ overhangs for complementary assembly.
  • a polynucleotide disclosed herein e.g., a RNA, e.g., an mRNA
  • a polynucleotide disclosed herein can be chemically synthesized using chemical synthesis methods and potential nucleobase substitutions known in the art.
  • the polynucleotides of the present invention e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide
  • their expression products, as well as degradation products and metabolites can be quantified according to methods known in the art.
  • the polynucleotides of the present invention can be quantified in exosomes or when derived from one or more bodily fluid.
  • peripheral blood serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood.
  • CSF cerebrospinal fluid
  • saliva aqueous humor
  • amniotic fluid cerumen
  • breast milk broncheoalveolar lavage fluid
  • semen prostatic fluid
  • exosomes can be retrieved from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.
  • exosome quantification method a sample of not more than 2mL is obtained from the subject and the exosomes isolated by size exclusion Attorney Docket No.45817-0124WO1 / MTX959.20 chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof.
  • the level or concentration of a polynucleotide can be an expression level, presence, absence, truncation or alteration of the administered construct. It is advantageous to correlate the level with one or more clinical phenotypes or with an assay for a human disease biomarker.
  • the assay can be performed using construct specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof while the exosomes can be isolated using immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods.
  • ELISA enzyme linked immunosorbent assay
  • Exosomes can also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof. [00471] These methods afford the investigator the ability to monitor, in real time, the level of polynucleotides remaining or delivered. This is possible because the polynucleotides of the present invention differ from the endogenous forms due to the structural or chemical modifications. [00472] In some embodiments, the polynucleotide can be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV/Vis).
  • UV/Vis ultraviolet visible spectroscopy
  • a non-limiting example of a UV/Vis spectrometer is a NANODROP® spectrometer (ThermoFisher, Waltham, MA).
  • the quantified polynucleotide can be analyzed in order to determine if the polynucleotide can be of proper size, check that no degradation of the polynucleotide has occurred.
  • Degradation of the polynucleotide can be checked by methods such as, but not limited to, agarose gel electrophoresis, HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE). Attorney Docket No.45817-0124WO1 / MTX959.20 20.
  • Pharmaceutical Compositions and Formulations [00473] The present invention provides pharmaceutical compositions and formulations that comprise any of the polynucleotides described above.
  • the composition or formulation further comprises a delivery agent.
  • the composition or formulation can contain a polynucleotide comprising a sequence optimized nucleic acid sequence disclosed herein which encodes a Snu13 polypeptide.
  • the composition or formulation can contain a polynucleotide comprising a sequence optimized nucleic acid sequence disclosed herein which encodes a Snu13 polypeptide and a polynucleotide comprising a sequence optimized nucleic acid sequence comprising a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500) and an ORF encoding a second polypeptide.
  • a polynucleotide comprising a sequence optimized nucleic acid sequence disclosed herein which encodes a Snu13 polypeptide and a polynucleotide comprising a sequence optimized nucleic acid sequence comprising a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500) and an ORF encoding a second polypeptide.
  • the composition or formulation can contain a polynucleotide (e.g., a RNA, e.g., an mRNA) comprising a polynucleotide (e.g., an ORF) having significant sequence identity to a sequence optimized nucleic acid sequence disclosed herein which encodes a Snu13polypeptide.
  • the polynucleotide further comprises a miRNA binding site, e.g., a miRNA binding site that binds miR-126, miR-142, miR-144, miR-146, miR-150, miR-155, miR-16, miR-21, miR-223, miR- 24, miR-27 and miR-26a.
  • compositions or formulation can optionally comprise one or more additional active substances, e.g., therapeutically and/or prophylactically active substances.
  • Pharmaceutical compositions or formulation of the present invention can be sterile and/or pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents can be found, for example, in Remington: The Science and Practice of Pharmacy 21 st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).
  • compositions are administered to humans, human patients or subjects.
  • the phrase "active ingredient" generally refers to polynucleotides to be delivered as described herein.
  • Formulations and pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In Attorney Docket No.45817-0124WO1 / MTX959.20 general, such preparatory methods include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit. [00477] A pharmaceutical composition or formulation in accordance with the present disclosure can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a "unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure can vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered.
  • the compositions and formulations described herein can contain at least one polynucleotide of the invention.
  • the composition or formulation can contain 1, 2, 3, 4 or 5 polynucleotides of the invention.
  • the compositions or formulations described herein can comprise more than one type of polynucleotide.
  • the composition or formulation can comprise a polynucleotide in linear and circular form.
  • the composition or formulation can comprise a circular polynucleotide and an in vitro transcribed (IVT) polynucleotide.
  • composition or formulation can comprise an IVT polynucleotide, a chimeric polynucleotide and a circular polynucleotide.
  • the present invention provides pharmaceutical formulations that comprise one or more polynucleotides described herein (e.g., one or more polynucleotides comprising nucleotide sequences encoding a Snu13 polypeptide). In some instances, the present invention provides pharmaceutical formulations that comprise a polynucleotide described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide).
  • the polynucleotides described herein can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation of the polynucleotide); (4) alter the biodistribution (e.g., target the polynucleotide to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein in vivo.
  • excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation of the polynucleotide); (4) alter the biodistribution (e.g., target the polynucleotide to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein in vivo.
  • the pharmaceutical formulation further comprises a delivery agent comprising, e.g., a compound having the Formula (I), e.g., Compound II or Compound B; or a compound having the Formula (III), (IV), (V), or (VI), e.g., Compound I or VI, or any combination thereof.
  • a delivery agent comprising, e.g., a compound having the Formula (I), e.g., Compound II or Compound B; or a compound having the Formula (III), (IV), (V), or (VI), e.g., Compound I or VI, or any combination thereof.
  • the delivery agent comprises an ionizable amino lipid (e.g., Compound II, VI, or B), a helper lipid (e.g., DSPC), a sterol (e.g., Cholesterol), and a PEG lipid (e.g., Compound I or PEG- DMG), e.g., with a mole ratio in the range of about (i) 40-50 mol% ionizable amino lipid (e.g., Compound II, VI, or B), optionally 45-50 mol% ionizable amino lipid, for example, 45-46 mol%, 46-47 mol%, 47-48 mol%, 48-49 mol%, or 49-50 mol% for example about 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol%, 48.5 mol%, 49 mol%, or 49.5 mol%; (ii) 30-45
  • the delivery agent comprises Compound B, Cholesterol, DSPC, and Compound I.
  • a pharmaceutically acceptable excipient includes, but are not limited to, any and all solvents, dispersion media, or other liquid vehicles, dispersion or suspension aids, diluents, granulating and/or dispersing agents, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, binders, lubricants or oil, coloring, sweetening or flavoring agents, stabilizers, antioxidants, antimicrobial or antifungal agents, osmolality adjusting agents, pH adjusting agents, buffers, chelants, cyoprotectants, and/or bulking agents, as suited to the particular dosage form desired.
  • Exemplary diluents include, but are not limited to, calcium or sodium carbonate, calcium phosphate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, etc., and/or combinations thereof.
  • Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], glyceryl monooleate, polyoxyethylene esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [BRIJ®30]), PLUORINC®F 68, POLOXAMER®188, etc.
  • natural emulsifiers e.g., acacia, a
  • Exemplary binding agents include, but are not limited to, starch, gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol), amino acids (e.g., glycine), natural and synthetic gums (e.g., acacia, sodium alginate), ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, etc., and combinations thereof.
  • Oxidation is a potential degradation pathway for mRNA, especially for liquid mRNA formulations.
  • antioxidants can be added Attorney Docket No.45817-0124WO1 / MTX959.20 to the formulations.
  • Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, m-cresol, methionine, butylated hydroxytoluene, monothioglycerol, sodium or potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, etc., and combinations thereof.
  • Exemplary chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, trisodium edetate, etc., and combinations thereof.
  • EDTA ethylenediaminetetraacetic acid
  • citric acid monohydrate disodium edetate
  • fumaric acid malic acid
  • phosphoric acid sodium edetate
  • tartaric acid trisodium edetate, etc.
  • Exemplary antimicrobial or antifungal agents include, but are not limited to, benzalkonium chloride, benzethonium chloride, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, benzoic acid, hydroxybenzoic acid, potassium or sodium benzoate, potassium or sodium sorbate, sodium propionate, sorbic acid, etc., and combinations thereof.
  • Exemplary preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, ascorbic acid, butylated hydroxyanisol, ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), etc., and combinations thereof.
  • the pH of polynucleotide solutions is maintained between pH 5 and pH 8 to improve stability.
  • Exemplary buffers to control pH can include, but are not limited to sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium malate, sodium carbonate, etc., and/or combinations thereof.
  • Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium or magnesium lauryl sulfate, etc., and combinations thereof.
  • the pharmaceutical composition or formulation described here can contain a cryoprotectant to stabilize a polynucleotide described herein during freezing.
  • Exemplary cryoprotectants include, but are not limited to mannitol, sucrose, trehalose, lactose, glycerol, dextrose, etc., and combinations thereof.
  • the pharmaceutical composition or formulation described here can contain a bulking agent in lyophilized polynucleotide formulations to yield a "pharmaceutically elegant" cake, stabilize the lyophilized polynucleotides during long term (e.g., 36 month) storage.
  • exemplary bulking agents of the present invention can include, but are not limited to sucrose, trehalose, mannitol, glycine, lactose, raffinose, and combinations thereof.
  • the pharmaceutical composition or formulation further comprises a delivery agent.
  • the delivery agent of the present disclosure can include, without limitation, liposomes, lipid nanoparticles, lipidoids, polymers, lipoplexes, microvesicles, exosomes, peptides, proteins, cells transfected with polynucleotides, hyaluronidase, nanoparticle mimics, nanotubes, conjugates, and combinations thereof.
  • Delivery Agents a. Lipid Compound [00495] The present disclosure provides pharmaceutical compositions with advantageous properties.
  • the lipid compositions described herein may be advantageously used in lipid nanoparticle compositions for the delivery of therapeutic and/or prophylactic agents, e.g., mRNAs, to mammalian cells or organs.
  • the lipids described herein have little or no immunogenicity.
  • the lipid compounds disclosed herein have a lower immunogenicity as compared to a reference lipid (e.g., MC3, KC2, or DLinDMA).
  • a formulation comprising a lipid disclosed herein and a therapeutic or prophylactic agent, e.g., mRNA has an increased therapeutic index as compared to a corresponding formulation which comprises a reference lipid (e.g., MC3, KC2, or DLinDMA) and the same therapeutic or prophylactic agent.
  • compositions comprising: (a) a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide; and (b) a delivery agent.
  • compositions comprising: (a) a first polynucleotide comprising a first nucleotide sequence encoding a Snu13 polypeptide, and a second polynucleotide comprising a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500) and an ORF encoding a second polypeptide (e.g., a target polypeptide); and (b) a delivery agent.
  • a first polynucleotide comprising a first nucleotide sequence encoding a Snu13 polypeptide
  • a second polynu13-binding site e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500
  • an ORF encoding a second polypeptide (e.g., a target polypeptide)
  • the present application provides: (a) a first pharmaceutical compositions comprising: (i) a first polynucleotide comprising a first nucleotide sequence encoding a Snu13 polypeptide, and (ii) a delivery agent; and (b) a second pharmaceutical composition comprising: (a second polynucleotide comprising a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500) and an ORF encoding a second polypeptide (e.g., a target polypeptide); and (i) a delivery agent.
  • a first pharmaceutical compositions comprising: (i) a first polynucleotide comprising a first nucleotide sequence encoding a Snu13 polypeptide, and (ii) a delivery agent
  • a second pharmaceutical composition comprising: (a second polynucleotide comprising a Sn
  • nucleic acids of the invention are formulated in a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • Lipid nanoparticles typically comprise ionizable cationic lipid, non-cationic lipid, sterol and PEG lipid components along with the nucleic acid cargo of interest.
  • the lipid nanoparticles of the invention can be generated using components, compositions, and methods as are generally known in the art, see for example PCT/US2016/052352; PCT/US2016/068300; PCT/US2017/037551; PCT/US2015/027400; PCT/US2016/047406; PCT/US2016000129; PCT/US2016/014280; PCT/US2016/014280; PCT/US2017/038426; PCT/US2014/027077; PCT/US2014/055394; PCT/US2016/52117; PCT/US2012/069610; PCT/US2017/027492; PCT/US2016/059575 and PCT/US2016/069491 all of which are incorporated by reference herein in their entirety.
  • Nucleic acids of the present disclosure are typically formulated in lipid nanoparticle.
  • the lipid nanoparticle comprises at least one ionizable cationic lipid, at least one non-cationic lipid, at least one sterol, and/or at least one polyethylene glycol (PEG)-modified lipid.
  • the lipid nanoparticle comprises a molar ratio of 20-60% ionizable cationic lipid.
  • the lipid nanoparticle may comprise a molar ratio of 40-50 mol%, optionally 45-50 mol%, for example, 45-46 mol%, 46-47 mol%, 47-48 mol%, 48-49 mol%, or 49-50 mol%, for example about 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol%, 48.5 mol%, 49 mol%, or 49.5 mol% ionizable cationic lipid.
  • the lipid nanoparticle comprises a molar ratio of 5- 25% non-cationic lipid.
  • the lipid nanoparticle may comprise a molar ratio of 5-15 mol%, optionally 10-12 mol%, for example, 5-6 mol%, 6-7 mol%, 7-8 mol%, 8-9 mol%, 9-10 mol%, 10-11 mol%, 11-12 mol%, 12-13 mol%, 13-14 mol%, or 14-15 mol% non-cationic lipid.
  • the lipid nanoparticle comprises a molar ratio of 25-55% sterol.
  • the lipid nanoparticle may comprise a molar ratio of 30- 45 mol%, optionally 35-40 mol%, for example, 30-31 mol%, 31-32 mol%, 32-33 mol%, 33-34 mol%, 35-35 mol%, 35-36 mol%, 36-37 mol%, 38-38 mol%, 38-39 mol%, or 39-40 mol% sterol.
  • the lipid nanoparticle comprises a molar ratio of 0.5-15% PEG-modified lipid.
  • the lipid nanoparticle may comprise a molar ratio of 1-5%, optionally 1-3 mol%, for example 1.5 to 2.5 mol%, 1-2 mol%, 2- 3 mol%, 3-4 mol%, or 4-5 mol% PEG-modified lipid.
  • the lipid nanoparticle comprises a molar ratio of 20-60% ionizable cationic lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5- 15% PEG-modified lipid.
  • the lipid nanoparticle comprises a molar ratio of 40-50% ionizable cationic lipid, 5-15% non-cationic lipid, 30-45% sterol, and 1-5% PEG-modified lipid.
  • the lipid nanoparticle comprises a molar ratio of 45-50% ionizable cationic lipid, 10-12% non-cationic lipid, 35-40% sterol, and 1-3% PEG-modified lipid.
  • the lipid nanoparticle comprises a molar ratio of 45-50% ionizable cationic lipid, 10-12% non-cationic lipid, 35-40% sterol, and 1.5- 2.5% PEG-modified lipid.
  • the disclosure relates to a compound of Formula (I): or its N-oxide, or a salt or isomer thereof, R’ branched denotes a point of attachment; wherein selected from the group consisting of H, C2-12 alkyl, and C2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C 1-14 alkyl and C 2-14 alkenyl; R 4 is selected from the group consisting of -(CH2)nOH, wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, , wherein denotes a point of attachment; wherein each R is independently selected from the group consisting of C 1-6 alkyl, C 2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Attorney Docket No.45817-0124WO1 / MTX959.20 each R 5 is independently selected from the group consisting of
  • R’ a is R’ branched ; denotes a point of attachment; R a ⁇ , R a ⁇ , R a ⁇ , C1-14 alkyl; R 4 is -(CH2)nOH; n is 2; and m is 7.
  • R’ a is R’ branched ; denotes a point of attachment; R a ⁇ , R a ⁇ , R a ⁇ , C 1-14 alkyl; R 4 is -(CH 2 ) n OH; n is 2; and m is 7.
  • R’ a is R’ branched ; denotes a point of attachment; R a ⁇ is C2-12 R 3 are each C1-14 alkyl; R 4 is Attorney Docket No.45817-0124WO1 / MTX959.20 6 alkyl); n2 is 2; R 5 is H; each R 6 is H; M and M’ are l is 5; and m is 7.
  • R’ a is R’ branched ; a point of attachment; R a ⁇ , R a ⁇ , and each C 1-14 alkyl; R 4 is - (CH2) n are each -C(O)O-; R’ is a C1- 12 alkyl; l is 5; and m is 7.
  • the compound of Formula (I) is selected from: . Attorney Docket No.45817-0124WO1 / MTX959.20 [00516]
  • the compound of Formula (I) is: . [00517] In is: . [00518] In is: (Compound B).
  • the disclosure relates to a compound of Formula (Ia): its N-oxide, or a salt or isomer thereof, R’ branched denotes a point of attachment; wherein selected from the group consisting of H, C2-12 alkyl, and C2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C 1-14 alkyl and C 2-14 alkenyl; Attorney Docket No.45817-0124WO1 / MTX959.20 R 4 is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, , wherein denotes a point R 10 is N each R is independently selected from the group consisting of 6 C 2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R 5 is independently selected from the group consisting of C 1-3 alkyl, C2-3 alkenyl, and
  • the disclosure relates to a compound of Formula (Ib): or its N-oxide, or a salt or isomer thereof, R’ branched denotes a point of attachment; wherein selected from the group consisting of H, C 2-12 alkyl, and C 2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl; Attorney Docket No.45817-0124WO1 / MTX959.20 R 4 is -(CH2)nOH, wherein n is selected from the group consisting of 1, 2, 3, 4, and 5; each R 5 is independently selected from the group consisting of C1-3 alkyl, C 2-3 alkenyl, and H; each R 6 is independently selected from the group consisting of C1-3 alkyl, C 2-3 alkenyl, and H; M and M’ are each independently selected from the group consisting of - C(O)O- and -OC(O)-; R’ is a C1-12 alky
  • R’ a is R’ branched ;
  • R’ branched is denotes a point of attachment;
  • R a ⁇ , R a ⁇ , and R a ⁇ are each H; alkyl;
  • R 4 is -(CH ) OH;
  • n is 2;
  • each R is H;
  • M and M’ are each -C(O)O-;
  • R’ is a C 1-12 alkyl; l is 5; and m is 7.
  • R’ a is R’ branched ;
  • R’ branched is denotes a point of attachment;
  • R a ⁇ , R a ⁇ , and R a ⁇ are each H; alkyl;
  • R 4 is -(CH 2 ) n OH; n is 2;
  • each R 5 is H;
  • each R 6 is H;
  • M and M’ are each -C(O)O-;
  • R’ is a C1-12 alkyl; l is 3; and m is 7.
  • R’ a is R’ branched ; R’ branched is a point of attachment; R a ⁇ and R a ⁇ are each H; R a ⁇ is C1-14 alkyl; R 4 is -(CH2)nOH; n is 2; each R 5 is H; each R 6 is H; M and M’ are each -C(O)O-; R’ is a C1-12 alkyl; l is 5; and m is 7.
  • the disclosure relates to a compound of Formula (Ic): Attorney Docket No.45817-0124WO1 / MTX959.20 its N-oxide, or a salt or isomer thereof, R’ branched denotes a point of attachment; wherein selected from the group consisting of H, C 2-12 alkyl, and C 2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl; , a point of attachment; wherein R is independently selected from the group consisting of C 1-6 alkyl, C 2-3 alkenyl, and H; n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R 5 is independently selected from the group consisting of C 1-3 alkyl, C2-3 alkenyl, and H; each R 6 is independently selected from the group consisting of C 1-3 alkyl, C2-3 alkenyl, and H; M and M
  • R’ a is R’ branched ; ; denotes a point of attachment; R a ⁇ , R a ⁇ , and R a ⁇ are alkyl; R 2 and R 3 are each C 1-14 alkyl; R 4 denotes a point of attachment; R 10 is NH(C1-6 alkyl); R 6 is H; M and M’ are each -C(O)O-; R’ is a C 1-12 alkyl; l is 5; and m is 7.
  • the compound of Formula (Ic) is: (Compound A).
  • R a ⁇ and R a ⁇ are each independently selected from the group consisting of H, C 1-12 alkyl, and C 2-12 alkenyl, wherein at least one of R a ⁇ and R a ⁇ is selected from the group consisting of C1-12 alkyl and C2-12 alkenyl;
  • R b ⁇ and R b ⁇ are each independently selected from the group consisting of H, C1-12 alkyl, and C2-12 alkenyl, wherein at least one of R b ⁇ and R b ⁇ is selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl;
  • R 2 and R 3 are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl;
  • R 4 is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, , wherein de
  • the disclosure relates to a compound of Formula (II-a): its N-oxide, or a salt or isomer thereof, Attorney Docket No.45817-0124WO1 / MTX959.20 ;
  • R a ⁇ and R a ⁇ are each independently selected from the group consisting of H, C1-12 alkyl, and C2-12 alkenyl, wherein at least one of R a ⁇ and R a ⁇ is selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl;
  • R b ⁇ and R b ⁇ are each independently selected from the group consisting of H, C 1-12 alkyl, and C 2-12 alkenyl, wherein at least one of R b ⁇ and R b ⁇ is selected from the group consisting of C1-12 alkyl and C2-12 alkenyl;
  • R 2 and R 3 are each independently selected from the group consisting of C 1-14 alkyl and C 2-14 alkenyl;
  • R 4 is selected from the group consisting of
  • the disclosure relates to a compound of Formula (II-c): its N-oxide, or a salt or isomer thereof, ; denotes a point of attachment; Attorney Docket No.45817-0124WO1 / MTX959.20 wherein R a ⁇ is selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl; R 4 is selected from the group consisting of -(CH 2 ) n OH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, , wherein denotes a point R 10 is N each R is independently selected from the group consisting of C 1-6 alkyl, C 2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; R’ is a C 1-12 alkyl or C 2-12 alken
  • the disclosure relates to a compound of Formula (II-d): ; wherein R a ⁇ and R b ⁇ are each independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; Attorney Docket No.45817-0124WO1 / MTX959.20
  • R 4 is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, , wherein denotes a point R 10 is N
  • each R is independently selected from the group consisting of 6 C2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R’ independently is a C1-12 alkyl or C2-12 alkenyl;
  • m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;
  • l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.
  • the disclosure relates to a compound of Formula (II-e): its N-oxide, or a salt or isomer thereof, ; selected from the group consisting of C1-12 alkyl and C2-12 R 2 and R 3 are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl; R 4 is -(CH2)nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5; R’ is a C1-12 alkyl or C2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.
  • m and l are each independently selected from 4, 5, and 6.
  • m and l are each 5.
  • each R’ independently is a C 1-12 alkyl. In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), each R’ independently is a C2-5 alkyl.
  • R’ b is: and R 2 and R 3 are each independently a C1-14 alkyl.
  • R’ b is: and R 2 and R 3 are each independently a C 6-10 alkyl.
  • R 3 are each Formula (II), is: Attorney Docket No.45817-0124WO1 / MTX959.20 [00536] In some embodiments of the compound of Formula (II), (II-a), (II-b), (II- is: b), (II- , , or , m are and each
  • m and l are each 5 and each R’ independently is a C 2-5 alkyl.
  • R’ branched l are each independently alkyl, and R a ⁇ and R b ⁇ are each a C 1-12 alkyl.
  • R’ b is: , m and l are each 5, each R’ independently is a C2-5 alkyl, and R a ⁇ and R b ⁇ are each a C 2-6 alkyl.
  • R’ branched are each independently selected and R 2 and R 3 are each independently a C 6-10 alkyl.
  • R’ branched is: Attorney Docket No.45817-0124WO1 / MTX959.20 l are each 5, R’ is a C 2-5 alkyl, R a ⁇ is a of Formula (II), (II-a), (II-b), (II- , wherein R 10 is NH(C 1-6 alkyl) and n2 is Formula (II), (II-a), (II-b), (II-c), (II-d), , wherein R 10 is NH(CH3) and n2 is 2.
  • R’ independently is a C2-5 alkyl, R a ⁇ and 6 alkyl, and R 4 , wherein R 10 is NH(CH 3 ) and embodiments of the compound of Formula (II), (II-a), (II-b), (II- c), (II-d), or (II-e), R’ branched are Attorney Docket No.45817-0124WO1 / MTX959.20 each independently selected from 4, 5, and 6, R’ is a C1-12 alkyl, R 2 and R 3 are each independently a C6-10 alkyl, R a ⁇ is a C1-12 alkyl, and R 4 , wherein R 10 is NH(C 1-6 alkyl) and n2 is 2.
  • each a C 8 alkyl, and R 4 wherein R 10 is NH(CH 3 ) and n2 is 2.
  • R 4 is -(CH2)nOH and n is 2, 3, or 4.
  • R 4 is -(CH 2 ) n OH and n is 2.
  • n is 2.
  • the disclosure relates to a compound of Formula (II-f): Attorney Docket No.45817-0124WO1 / MTX959.20 alkyl; each independently a C1-14 alkyl; is -(CH 2 ) n OH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5; R’ is a C 1-12 alkyl; m is selected from 4, 5, and 6; and l is selected from 4, 5, and 6. [00546] In some embodiments of the compound of Formula (II-f), m and l are each 5, and n is 2, 3, or 4.
  • R’ is a C2-5 alkyl, R a ⁇ is a C 2-6 alkyl, and R 2 and R 3 are each a C 6-10 alkyl.
  • m and l are each 5, n is 2, 3, or 4
  • R’ is a C 2-5 alkyl, R a ⁇ is a C 2-6 alkyl, and R 2 and R 3 are each a C 6-10 alkyl.
  • the disclosure relates to a compound of Formula (II-g):
  • R 4 is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting of 3, 4, and 5, , wherein denotes a point of (C 1-6 alkyl), and n2 is selected from the group consisting of 1, 2, and 3.
  • a 5 R 4 is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting of 3, 4, and 5, , wherein denotes a point of , and n2 is selected from consisting of 1, 2, and 3.
  • R 4 is , wherein n2 is 2. [00552] In some embodiments of the compound of Formula (II-g) or (II-h), R 4 is - (CH 2 ) 2 OH.
  • the disclosure relates to a compound having the Formula (III): Attorney Docket No.45817-0124WO1 / MTX959.20 , or a salt or R 1 , consisting of C5-20 alkyl, C5-20 alkenyl, -R”MR’, -R*YR”, -YR”, and -R*OR”; each M is independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, an aryl group, and a heteroaryl group; X 1 , X 2 , and X 3 are
  • R1, R2, R3, R4, and R5 are each C5-20 alkyl; X 1 is -CH2-; and X 2 and X 3 are each -C(O)-.
  • the compound of Formula (III) is: Attorney Docket No.45817-0124WO1 / MTX959.20 a [00556]
  • the lipid composition of the lipid nanoparticle composition disclosed herein can comprise one or more phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.
  • a phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.
  • a fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
  • Particular phospholipids can facilitate fusion to a membrane.
  • a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.
  • a lipid-containing composition e.g., LNPs
  • Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated.
  • a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond).
  • alkynes e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond.
  • an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide.
  • Such reactions can be useful in functionalizing a lipid bilayer of Attorney Docket No.45817-0124WO1 / MTX959.20 a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).
  • a targeting or imaging moiety e.g., a dye
  • Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.
  • a phospholipid of the invention comprises 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero- 3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero
  • a phospholipid useful or potentially useful in the present invention is an analog or variant of DSPC.
  • a phospholipid useful or potentially useful in the present invention is a compound of Formula (IV): Attorney Docket No.45817-0124WO1 / MTX959.20 or a salt thereof, wherein: each R 1 is independently optionally substituted alkyl; or optionally two R 1 are joined together with the intervening atoms to form optionally substituted monocyclic carbocyclyl or optionally substituted monocyclic heterocyclyl; or optionally three R 1 are joined together with the intervening atoms to form optionally substituted bicyclic carbocyclyl or optionally substitute bicyclic heterocyclyl; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is of the ; each instance of L 2 substituted C1-6 alkylene, wherein one methylene unit of the optionally substituted C 1-6
  • a phospholipid useful or potentially useful in the present invention comprises a modified phospholipid head (e.g., a modified choline group).
  • a phospholipid with a modified head is DSPC, or analog thereof, with a modified quaternary amine.
  • at least one of R 1 is not methyl. In certain embodiments, at least one of R 1 is not hydrogen or methyl.
  • the compound of Formula (IV) is of one of the following Formulae: , each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each u is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and each v is independently 1, 2, or 3.
  • a compound of Formula (IV) is of Formula (IV-a): Attorney Docket No.45817-0124WO1 / MTX959.20 or a salt thereof.
  • a phospholipid useful or potentially useful in the present invention comprises a cyclic moiety in place of the glyceride moiety.
  • a phospholipid useful in the present invention is DSPC, or analog thereof, with a cyclic moiety in place of the glyceride moiety.
  • the compound of Formula (IV) is of Formula (IV-b): , or a salt thereof.
  • Phospholipid Tail Modifications [00567]
  • a phospholipid useful or potentially useful in the present invention comprises a modified tail.
  • a phospholipid useful or potentially useful in the present invention is DSPC, or analog thereof, with a modified tail.
  • a “modified tail” may be a tail with shorter or longer aliphatic chains, aliphatic chains with branching introduced, aliphatic chains with substituents introduced, aliphatic chains wherein one or more methylenes are replaced by cyclic or heteroatom groups, or any combination thereof.
  • the compound of (IV) is of Formula (IV-a), or a salt thereof, wherein at least one instance of R 2 is each instance of R 2 is optionally substituted C1- 30 alkyl, wherein one or more methylene units of R 2 are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, - C(O), C(O)N(R N ), NR N C(O), NR N C(O)N(R N ), C(O)O, OC(O), OC(O)O, - , Attorney Docket No.45817-0124WO1 / MTX959.20 [00568]
  • the compound of Formula (IV) is of Formula (IV- c): c), or a salt thereof, wherein: each x is each instance is G is independently selected from the group consisting of optionally substitute
  • a phospholipid useful or potentially useful in the present invention comprises a modified phosphocholine moiety, wherein the alkyl chain linking the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Therefore, in certain embodiments, a phospholipid useful or potentially useful in the present invention is a compound of Formula (IV), wherein n is 1, 3, 4, 5, 6, 7, 8, 9, or 10.
  • a compound of Formula (IV) is of one of the following Formulae: , or a salt Attorney Docket No.45817-0124WO1 / MTX959.20
  • Alternative Lipids [00570]
  • a phospholipid useful or potentially useful in the present invention comprises a modified phosphocholine moiety, wherein the alkyl chain linking the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Therefore, in certain embodiments, a phospholipid useful.
  • an alternative lipid is used in place of a phospholipid of the present disclosure.
  • an alternative lipid of the invention is oleic acid.
  • the alternative lipid is one of the following: , , , , Attorney Docket No.45817-0124WO1 / MTX959.20
  • the lipid composition of a pharmaceutical composition disclosed herein can comprise one or more structural lipids.
  • structural lipid refers to sterols and also to lipids containing sterol moieties.
  • Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle.
  • Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof.
  • the structural lipid is a sterol.
  • sterols are a subgroup of steroids consisting of steroid alcohols.
  • the structural lipid is a steroid.
  • the structural lipid is cholesterol.
  • the structural lipid is an analog of cholesterol.
  • the structural lipid is alpha-tocopherol.
  • the structural lipids may be one or more of the structural lipids described in U.S. Application No.62/520,530.
  • Polyethylene Glycol (PEG)-Lipids [00577]
  • the lipid composition of a pharmaceutical composition disclosed herein can comprise one or more a polyethylene glycol (PEG) lipid.
  • PEG-lipid refers to polyethylene glycol (PEG)- modified lipids.
  • Non-limiting examples of PEG-lipids include PEG-modified Attorney Docket No.45817-0124WO1 / MTX959.20 phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2- diacyloxypropan-3-amines.
  • PEGylated lipids Such lipids are also referred to as PEGylated lipids.
  • a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • the PEG-lipid includes, but not limited to 1,2- dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG- disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG- diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG- DPPE), or PEG-l,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).
  • PEG-DMG 1,2- dimyristoyl-sn-glycerol methoxypolyethylene glycol
  • PEG-DSPE 1,2-distearoyl-sn- g
  • the PEG-lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.
  • the lipid moiety of the PEG-lipids includes those having lengths of from about C 14 to about C 22 , preferably from about C 14 to about C 16 .
  • a PEG moiety for example an mPEG-NH2 has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons.
  • the PEG- lipid is PEG2k-DMG.
  • the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG.
  • Non-limiting examples of non-diffusible PEGs include PEG-DSG and PEG-DSPE.
  • PEG-lipids are known in the art, such as those described in U.S. Patent No. 8158601 and International Publ. No. WO 2015/130584 A2, which are incorporated herein by reference in their entirety.
  • lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids.
  • a PEG lipid is a lipid modified with polyethylene glycol.
  • a PEG lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamines, PEG- modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof.
  • a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • the PEG-modified lipids are a modified form of PEG DMG.
  • PEG-DMG has the following structure: [00587]
  • PEGylated lipids described in International Publication No. WO2012099755 the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain.
  • the PEG lipid is a PEG-OH lipid.
  • a “PEG-OH lipid” (also referred to herein as “hydroxy- PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (–OH) groups on the lipid.
  • the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain.
  • a PEG-OH or hydroxy-PEGylated lipid comprises an –OH group at the terminus of the PEG chain.
  • a PEG lipid useful in the present invention is a compound of Formula (V).
  • R 3 is –OR O ;
  • R O is hydrogen, optionally substituted alkyl, or an oxygen protecting group;
  • r is an integer between 1 and 100, inclusive;
  • L 1 is optionally substituted C 1-10 alkylene, wherein at least one methylene of the optionally substituted C1-10 alkylene is independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(R N ), S, C(O), - C(O)N(R N ), NR N C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, or - NR N C N m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
  • A is of the
  • the compound of Formula (V) is a PEG-OH lipid (i.e., R 3 is –OR O , and R O is hydrogen).
  • the compound of Formula (V) is of Formula (V-OH): (V-OH), or a salt thereof.
  • a PEG lipid useful in the present invention is a compound of Formula (VI).
  • R O is hydrogen, optionally substituted alkyl or an oxygen protecting group
  • r is an integer between 1 and 100, inclusive
  • R 5 is optionally substituted C 10-40 alkyl, optionally substituted C 10-40 alkenyl, or optionally substituted C10-40 alkynyl
  • optionally one or more methylene groups of R 5 are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O)N(R N ), NR N C(O), NR N C(O)N(R N ), C(O)O, OC(O), - , - , - or a nitrogen protecting group.
  • the compound of Formula (VI) is of Formula (VI- OH): Attorney Docket No.45817-0124WO1 / MTX959.20 (VI-OH), or a salt thereof.
  • the compound of Formula (VI) is: . or a
  • the compound of Formula (VI) is some aspects, compositions disclosed herein does not comprise a PEG-lipid.
  • the PEG-lipids may be one or more of the PEG lipids described in U.S. Application No.62/520,530.
  • a PEG lipid of the invention comprises a PEG- modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG- modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.
  • the PEG-modified lipid is PEG-DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG and/or PEG-DPG.
  • a LNP of the invention comprises an ionizable cationic lipid of any of Formula I, II or III, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising PEG-DMG.
  • a LNP of the invention comprises an ionizable cationic lipid of any of Formula I, II or III, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising a compound having Formula VI.
  • a LNP of the invention comprises an ionizable cationic lipid of Formula I, II or III, a phospholipid comprising a compound having Formula IV, a structural lipid, and the PEG lipid comprising a compound having Formula V or VI.
  • a LNP of the invention comprises an ionizable cationic lipid of Formula I, II or III, a phospholipid comprising a compound having Formula IV, a structural lipid, and the PEG lipid comprising a compound having Formula V or VI.
  • a LNP of the invention comprises an ionizable cationic lipid of Formula I, II or III, a phospholipid having Formula IV, a structural lipid, and a PEG lipid comprising a compound having Formula VI.
  • a LNP of the invention comprises an ionizable cationic lipid of , [00603] In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of , [00604] In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of , lipid comprising cholesterol, and a PEG lipid comprising a compound having Formula VI.
  • a LNP of the invention comprises an ionizable cationic lipid of Attorney Docket No.45817-0124WO1 / MTX959.20 and a a [00606]
  • a LNP of the invention comprises an ionizable cationic lipid of a phospholipid comprising DOPE, a structural lipid comprising cholesterol, and a PEG lipid comprising a compound having Formula VI.
  • a LNP of the invention comprises an N:P ratio of from about 2:1 to about 30:1.
  • a LNP of the invention comprises an N:P ratio of about 6:1.
  • a LNP of the invention comprises an N:P ratio of about 3:1.
  • a LNP of the invention comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of from about 10:1 to about 100:1.
  • a LNP of the invention comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of about 20:1.
  • a LNP of the invention comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of about 10:1.
  • a LNP of the invention has a mean diameter from about 50nm to about 150nm.
  • a LNP of the invention has a mean diameter from about 70nm to about 120nm.
  • alkyl As used herein, the term “alkyl”, “alkyl group”, or “alkylene” means a linear or branched, saturated hydrocarbon including one or more carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms), which is optionally substituted.
  • C1-14 alkyl means an optionally substituted linear or branched, saturated hydrocarbon including 1-14 carbon atoms.
  • alkyl group described herein refers to both unsubstituted and substituted alkyl groups.
  • alkenyl means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one double bond, which is optionally substituted.
  • C 2-14 alkenyl means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon double bond.
  • An alkenyl group may include one, two, three, four, or more carbon-carbon double bonds.
  • C18 alkenyl may include one or more double bonds.
  • a C 18 alkenyl group including two double bonds may be a linoleyl group.
  • an alkenyl group described herein refers to both unsubstituted and substituted alkenyl groups.
  • alkynyl means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one carbon-carbon triple bond, which is optionally substituted.
  • C 2-14 alkynyl means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon triple bond.
  • An alkynyl group may include one, two, three, four, or more carbon-carbon triple bonds.
  • C18 alkynyl may include one or more carbon-carbon triple bonds.
  • an alkynyl group described herein refers to both unsubstituted and substituted alkynyl groups.
  • the term "carbocycle” or “carbocyclic group” means an optionally substituted mono- or multi-cyclic system including one or more rings of Attorney Docket No.45817-0124WO1 / MTX959.20 carbon atoms.
  • Rings may be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty membered rings.
  • C3-6 carbocycle means a carbocycle including a single ring having 3-6 carbon atoms.
  • Carbocycles may include one or more carbon- carbon double or triple bonds and may be non-aromatic or aromatic (e.g., cycloalkyl or aryl groups). Examples of carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and 1,2 dihydronaphthyl groups.
  • cycloalkyl as used herein means a non-aromatic carbocycle and may or may not include any double or triple bond. Unless otherwise specified, carbocycles described herein refers to both unsubstituted and substituted carbocycle groups, i.e., optionally substituted carbocycles. [00619] As used herein, the term “heterocycle” or “heterocyclic group” means an optionally substituted mono- or multi-cyclic system including one or more rings, where at least one ring includes at least one heteroatom. Heteroatoms may be, for example, nitrogen, oxygen, or sulfur atoms.
  • Rings may be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen membered rings.
  • Heterocycles may include one or more double or triple bonds and may be non- aromatic or aromatic (e.g., heterocycloalkyl or heteroaryl groups).
  • heterocycles include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, and isoquinolyl groups.
  • heterocycloalkyl as used herein means a non-aromatic heterocycle and may or may not include any double or triple bond. Unless otherwise specified, heterocycles described herein refers to both unsubstituted and substituted heterocycle groups, i.e., optionally substituted heterocycles.
  • heteroalkyl refers respectively to an alkyl, alkenyl, alkynyl group, as defined herein, which further comprises one or more (e.g., 1, 2, 3, or 4) heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus) wherein the one or more heteroatoms is inserted between adjacent carbon atoms within the parent carbon chain and/or one or more heteroatoms is inserted between a carbon atom and the parent Attorney Docket No.45817-0124WO1 / MTX959.20 molecule, i.e., between the point of attachment.
  • heteroatoms e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus
  • heteroalkyls, heteroalkenyls, or heteroalkynyls described herein refers to both unsubstituted and substituted heteroalkyls, heteroalkenyls, or heteroalkynyls, i.e., optionally substituted heteroalkyls, heteroalkenyls, or heteroalkynyls.
  • a "biodegradable group” is a group that may facilitate faster metabolism of a lipid in a mammalian entity.
  • a biodegradable group may be selected from the group consisting of, but is not limited to, -C(O)O-, -OC(O)-, - C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, - P(O)(OR')O-, -S(O)2-, an aryl group, and a heteroaryl group.
  • an "aryl group” is an optionally substituted carbocyclic group including one or more aromatic rings. Examples of aryl groups include phenyl and naphthyl groups.
  • heteroaryl group is an optionally substituted heterocyclic group including one or more aromatic rings.
  • heteroaryl groups include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, and thiazolyl. Both aryl and heteroaryl groups may be optionally substituted.
  • M and M' can be selected from the non- limiting group consisting of optionally substituted phenyl, oxazole, and thiazole. In the Formulas herein, M and M' can be independently selected from the list of biodegradable groups above.
  • aryl or heteroaryl groups described herein refers to both unsubstituted and substituted groups, i.e., optionally substituted aryl or heteroaryl groups.
  • Alkyl, alkenyl, and cyclyl (e.g., carbocyclyl and heterocyclyl) groups may be optionally substituted unless otherwise specified.
  • R is an alkyl or alkenyl group, as defined herein.
  • the substituent groups themselves may be further substituted with, for example, one, two, three, four, five, or six substituents as defined herein.
  • a C1-6 alkyl group may be further substituted with one, two, three, four, five, or six substituents as described herein.
  • N-hydroxy compounds can be prepared by oxidation of the parent amine by an oxidizing agent such as m CPBA.
  • the lipid composition of a pharmaceutical composition disclosed herein can include one or more components in addition to those described above.
  • the lipid composition can include one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents (e.g., surfactants), or other components.
  • a permeability enhancer molecule can be a molecule described by U.S. Patent Application Publication No.2005/0222064.
  • Carbohydrates Attorney Docket No.45817-0124WO1 / MTX959.20 can include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof).
  • a polymer can be included in and/or used to encapsulate or partially encapsulate a pharmaceutical composition disclosed herein (e.g., a pharmaceutical composition in lipid nanoparticle form).
  • a polymer can be biodegradable and/or biocompatible.
  • a polymer can be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.
  • the ratio between the lipid composition and the polynucleotide range can be from about 10:1 to about 60:1 (wt/wt).
  • the ratio between the lipid composition and the polynucleotide can be about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1 or 60:1 (wt/wt).
  • the wt/wt ratio of the lipid composition to the polynucleotide encoding a therapeutic agent is about 20:1 or about 15:1.
  • the pharmaceutical composition disclosed herein can contain more than one polypeptides.
  • a pharmaceutical composition disclosed herein can contain two or more polynucleotides (e.g., RNA, e.g., mRNA).
  • the lipid nanoparticles described herein can comprise polynucleotides (e.g., mRNA) in a lipid:polynucleotide weight ratio of 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1 or 70:1, or a range or any of these ratios such as, but not limited to, 5:1 to about 10:1, from about 5:1 to about 15:1, from about 5:1 to about 20:1, from about 5:1 to about 25:1, from about 5:1 to about 30:1, from about 5:1 to about 35:1, from about 5:1 to about 40:1, from about 5:1 to about 45:1, from about 5:1 to about 50:1, from about 5:1 to about 55:1, from about 5:1 to about 60:1, from about 5:1 to about 70:1, from about 10:1 to about 15:1, from about 10:1 to about 20:1, from about 10:
  • the lipid nanoparticles described herein can comprise the polynucleotide in a concentration from approximately 0.1 mg/ml to 2 mg/ml such as, but not limited to, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2.0 mg/ml or greater than 2.0 mg/ml.
  • Nanoparticle Compositions [00631]
  • the pharmaceutical compositions disclosed herein are formulated as lipid nanoparticles (LNP).
  • the present disclosure also provides nanoparticle compositions comprising (i) a lipid composition comprising a delivery agent such as compound as described herein, and (ii) a polynucleotide encoding a Snu13 polypeptide.
  • the present disclosure also provides nanoparticle compositions comprising (i) a lipid composition comprising a delivery agent such as compound as described herein, and (ii) a first polynucleotide encoding a Snu13 polypeptide and a second polynucleotide encoding a second polypeptide (e.g., a target polypeptide) and comprising a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500).
  • a delivery agent such as compound as described herein
  • a first polynucleotide encoding a Snu13 polypeptide and a second polynucleotide encoding a second polypeptide (e.g., a target polypeptide) and comprising a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521,
  • the lipid composition disclosed herein can encapsulate the polynucleotide(s) (e.g., the polynucleotide encoding the Snu13 polypeptide; or the polynucleotide encoding the Snu13 polypeptide and the second polynucleotide encoding the second (target) polypeptide).
  • Nanoparticle compositions are typically sized on the order of micrometers or smaller and can include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes.
  • Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, and lipoplexes.
  • LNPs lipid nanoparticles
  • nanoparticle compositions are vesicles including one or more lipid bilayers.
  • a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments.
  • Lipid bilayers can be functionalized and/or crosslinked to one another.
  • Lipid bilayers can include one or more ligands, proteins, or channels.
  • a lipid nanoparticle comprises an ionizable amino lipid, a structural lipid, a phospholipid, and mRNA.
  • the LNP comprises an ionizable amino lipid, a PEG-modified lipid, a sterol and a structural lipid.
  • the LNP has a molar ratio of about 40-50% ionizable amino lipid; about 5-15% structural lipid; about 30-45% sterol; and about 1-5% PEG- modified lipid.
  • the LNP has a polydispersity value of less than 0.4.
  • the LNP has a net neutral charge at a neutral pH.
  • the LNP has a mean diameter of 50-150 nm. In some embodiments, the LNP has a mean diameter of 80-100 nm.
  • the term “lipid” refers to a small molecule that has hydrophobic or amphiphilic properties. Lipids may be naturally occurring or synthetic. Examples of classes of lipids include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides, and prenol lipids.
  • a lipid nanoparticle may comprise an ionizable amino lipid.
  • the term “ionizable amino lipid” has its ordinary meaning in the art and may refer to a lipid comprising one or more charged moieties.
  • an ionizable amino lipid may be positively charged or negatively charged.
  • An ionizable amino lipid may be positively charged, in which case it can be referred to as “cationic lipid”.
  • an ionizable amino lipid molecule may comprise an amine group, and can be referred to as an Attorney Docket No.45817-0124WO1 / MTX959.20 ionizable amino lipid.
  • a “charged moiety” is a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1, or -1), divalent (+2, or -2), trivalent (+3, or -3), etc.
  • the charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged).
  • Examples of positively-charged moieties include amine groups (e.g., primary, secondary, and/or tertiary amines), ammonium groups, pyridinium group, guanidine groups, and imidizolium groups.
  • the charged moieties comprise amine groups.
  • Examples of negatively- charged groups or precursors thereof include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like.
  • the charge of the charged moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and/or cause the moiety to become charged or uncharged.
  • the charge density of the molecule may be selected as desired.
  • charge or “charged moiety” does not refer to a “partial negative charge” or “partial positive charge” on a molecule.
  • the terms “partial negative charge” and “partial positive charge” are given its ordinary meaning in the art.
  • a “partial negative charge” may result when a functional group comprises a bond that becomes polarized such that electron density is pulled toward one atom of the bond, creating a partial negative charge on the atom.
  • the ionizable amino lipid is sometimes referred to in the art as an “ionizable cationic lipid”.
  • the ionizable amino lipid may have a positively charged hydrophilic head and a hydrophobic tail that are connected via a linker structure.
  • an ionizable amino lipid may also be a lipid including a cyclic amine group.
  • the ionizable amino lipid may be selected from, but not limited to, an ionizable amino lipid described in International Publication Nos. WO2013086354 and WO2013116126; the contents of each of which are herein incorporated by reference in their entirety.
  • the ionizable amino lipid may be selected from, but not limited to, Formula CLI-CLXXXII of US Patent No.7,404,969; each of which is herein incorporated by reference in their entirety.
  • the lipid may be a cleavable lipid such as those described in International Publication No. WO2012170889, herein incorporated by reference in its entirety.
  • the lipid may be synthesized by methods known in the art and/or as described in International Publication Nos. WO2013086354; the contents of each of which are herein incorporated by reference in their entirety.
  • Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) can be used to measure zeta potentials. Dynamic light scattering can also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple characteristics of a nanoparticle composition, such as particle size, polydispersity index, and zeta potential.
  • microscopy e.g., transmission electron microscopy or scanning electron microscopy
  • Dynamic light scattering or potentiometry e.g., potentiometric titrations
  • Dynamic light scattering can also be utilized to determine particle sizes.
  • Instruments such as the Ze
  • the size of the nanoparticles can help counter biological reactions such as, but not limited to, inflammation, or can increase the biological effect of the polynucleotide.
  • size or mean size in the context of nanoparticle compositions refers to the mean diameter of a nanoparticle composition.
  • the polynucleotide encoding a Snu13 polypeptide is formulated in lipid nanoparticles having a diameter from about 10 to about 100 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 20 to about 100
  • the nanoparticles have a diameter from about 10 to 500 nm. In one embodiment, the nanoparticle has a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm.
  • the largest dimension of a nanoparticle composition is 1 ⁇ m or shorter (e.g., 1 ⁇ m, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or shorter).
  • a nanoparticle composition can be relatively homogenous.
  • a polydispersity index can be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition.
  • a small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution.
  • a nanoparticle composition can have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25.
  • the polydispersity index of a nanoparticle composition disclosed herein can be from about 0.10 to about 0.20.
  • the zeta potential of a nanoparticle composition can be used to indicate the electrokinetic potential of the composition.
  • the zeta potential can describe the surface charge of a nanoparticle composition.
  • Nanoparticle compositions with relatively low charges, positive or negative, are generally desirable, as more highly charged species can interact undesirably with cells, tissues, and other elements in the body.
  • the zeta potential of a nanoparticle composition Attorney Docket No.45817-0124WO1 / MTX959.20 disclosed herein can be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about 10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 m
  • the zeta potential of the lipid nanoparticles can be from about 0 mV to about 100 mV, from about 0 mV to about 90 mV, from about 0 mV to about 80 mV, from about 0 mV to about 70 mV, from about 0 mV to about 60 mV, from about 0 mV to about 50 mV, from about 0 mV to about 40 mV, from about 0 mV to about 30 mV, from about 0 mV to about 20 mV, from about 0 mV to about 10 mV, from about 10 mV to about 100 mV, from about 10 mV to about 90 mV, from about 10 mV to about 80 mV, from about 10 mV to about 70 mV, from about 10 mV to about 60 mV, from about 10 mV to about 50 mV, from about 10 mV to about 40 m
  • the zeta potential of the lipid nanoparticles can be from about 10 mV to about 50 mV, from about 15 mV to about 45 mV, from about 20 mV to about 40 mV, and from about 25 mV to about 35 mV. In some embodiments, the zeta potential of the lipid nanoparticles can be about 10 mV, about 20 mV, about 30 mV, Attorney Docket No.45817-0124WO1 / MTX959.20 about 40 mV, about 50 mV, about 60 mV, about 70 mV, about 80 mV, about 90 mV, and about 100 mV.
  • encapsulation efficiency of a polynucleotide describes the amount of the polynucleotide that is encapsulated by or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided.
  • encapsulation can refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement.
  • Encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of the polynucleotide in a solution containing the nanoparticle composition before and after breaking up the nanoparticle composition with one or more organic solvents or detergents.
  • the encapsulation efficiency of a polynucleotide can be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.
  • the amount of a polynucleotide present in a pharmaceutical composition disclosed herein can depend on multiple factors such as the size of the polynucleotide, desired target and/or application, or other properties of the nanoparticle composition as well as on the properties of the polynucleotide.
  • the amount of an mRNA useful in a nanoparticle composition can depend on the size (expressed as length, or molecular mass), sequence, and other characteristics of the mRNA.
  • the relative amounts of a polynucleotide in a nanoparticle composition can also vary.
  • the relative amounts of the lipid composition and the polynucleotide present in a lipid nanoparticle composition of the present disclosure can be optimized according to considerations of efficacy and tolerability.
  • the N:P ratio can serve as a useful metric.
  • nanoparticle compositions with low N:P ratios and strong expression Attorney Docket No.45817-0124WO1 / MTX959.20 are desirable.
  • N:P ratios vary according to the ratio of lipids to RNA in a nanoparticle composition.
  • a lower N:P ratio is preferred.
  • the one or more RNA, lipids, and amounts thereof can be selected to provide an N:P ratio from about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1.
  • the N:P ratio can be from about 2:1 to about 8:1.
  • the N:P ratio is from about 5:1 to about 8:1.
  • the N:P ratio is between 5:1 and 6:1.
  • the N:P ratio is about is about 5.67:1.
  • the present disclosure also provides methods of producing lipid nanoparticles comprising encapsulating a polynucleotide.
  • Such method comprises using any of the pharmaceutical compositions disclosed herein and producing lipid nanoparticles in accordance with methods of production of lipid nanoparticles known in the art. See, e.g., Wang et al. (2015) “Delivery of oligonucleotides with lipid nanoparticles” Adv. Drug Deliv. Rev.87:68- 80; Silva et al. (2015) “Delivery Systems for Biopharmaceuticals. Part I: Nanoparticles and Microparticles” Curr. Pharm. Technol.16: 940-954; Naseri et al.
  • mRNA-Lipid Adducts [00662] It has been determined that certain ionizable lipids are susceptible to the formation of lipid-polynucleotide adducts.
  • ionizable lipids that comprise a tertiary amine group may decompose into one or both of a secondary amine and a reactive aldehyde species capable of interacting with polynucleotides (such as mRNA) to form an ionizable lipid-polynucleotide adduct impurity that can be detected by reverse phase ion pair chromatography (RP-IP HPLC).
  • RP-IP HPLC reverse phase ion pair chromatography
  • the ionizable lipid-polynucleotide adduct impurity is an aldehyde-mRNA adduct impurity.
  • LNP compositions with a reduced content of ionizable lipid-polynucleotide adduct impurity such as wherein less than about 20%, less than about 10%, less than about 5%, or less than about 1%, of the mRNA is in the form of ionizable lipid-polynucleotide adduct impurity, as may be measured by RP-IP HPLC.
  • an LNP composition wherein less than about 10%, less than about 5%, or less than about 1%, of the mRNA is in the form of ionizable lipid-polynucleotide adduct impurity, including less than 10%, less than 5%, or less than 1%, as may be measured by RP-IP HPLC.
  • an amount of lipid aldehydes in the composition is less than about 50 ppm, including less than 50 ppm.
  • an amount of N-oxide compounds in the composition is less than about 50 ppm, including less than 50 ppm.
  • an amount of transition metals, such as Fe, in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of alkyl halide compounds in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of anhydride compounds in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of ketone compounds in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of conjugated diene compounds in the composition is less than about 50 ppm, including less than 50 ppm.
  • the composition is stable against the formation of ionizable lipid-polynucleotide adduct impurity.
  • an amount of ionizable lipid-polynucleotide adduct impurity in the composition increases at an average rate of less than about 2% per day when stored at a temperature of about 25 °C or below, including at an average rate of less than 2% per day.
  • an amount of ionizable lipid-polynucleotide adduct impurity in the composition increases Attorney Docket No.45817-0124WO1 / MTX959.20 at an average rate of less than about 0.5% per day when stored at a temperature of about 5 °C or below, including at an average rate of less than 0.5% per day.
  • an amount of ionizable lipid-polynucleotide adduct impurity in the composition increases at an average rate of less than about 0.5% per day when stored at a refrigerated temperature, optionally wherein the refrigerated temperature is about 5 °C.
  • Lipid vehicle (e.g., LNP) compositions with a reduced content of ionizable lipid-polynucleotide adduct impurity can be prepared by methods that inhibit formation of one or both of N-oxides and aldehydes. Such methods may comprise treating a composition comprising an ionizable lipid comprising a tertiary amine group to inhibit formation of one or both of N-oxides and aldehydes, such as by treating the composition with a reducing agent; treating the composition with a chelating agent; adjusting the pH of the composition; adjusting the temperature of the composition; and adjusting the buffer in the composition.
  • LNP ionizable lipid-polynucleotide adduct impurity
  • Such methods may comprise, prior to combining the ionizable lipid with a polynucleotide, one or more of treating the ionizable lipid with a scavenging agent; treating the ionizable lipid with a reductive treatment agent; treating the ionizable lipid with a reducing agent; treating the ionizable lipid with a chelating agent; treating the polynucleotide with a reducing agent; and treating the polynucleotide with a chelating agent.
  • the scavenging agent, reductive treatment agent, and/or reducing agent may be an agent that reacts with aldehyde, ketone, anhydride and/or diene compounds.
  • a scavenging agent may comprise one or more selected from (O-(2,3,4,5,6-Pentafluorobenzyl)hydroxylamine hydrochloride) (PFBHA), methoxyamine (e.g., methoxyamine hydrochloride), benzyloxyamine (e.g., benzyloxyamine hydrochloride), ethoxyamine (e.g., ethoxyamine hydrochloride), 4- [2-(aminooxy)ethyl]morpholine dihydrochloride, butoxyamine (e.g., tert-butoxyamine hydrochloride), 4-Dimethylaminopyridine (DMAP), 1,4-diazabicyclo[2.2.2]octane (DABCO), Triethylamine (TEA), Piperidine 4-carboxylate (BPPC), and combinations thereof.
  • PFBHA fluorobenzyl)hydroxylamine hydrochloride
  • methoxyamine e.g., methoxyamine hydroch
  • a reductive treatment agent may comprise a boron compound (e.g., sodium borohydride and/or bis(pinacolato)diboron).
  • a reductive treatment agent may comprise a boron compound, such as one or both of sodium borohydride and bis(pinacolato)diboron).
  • a chelating agent may comprise immobilized iminodiacetic Attorney Docket No.45817-0124WO1 / MTX959.20 acid.
  • a reducing agent may comprise an immobilized reducing agent, such as immobilized diphenylphosphine on silica (Si-DPP), immobilized thiol on agarose (Ag-Thiol), immobilized cysteine on silica (Si-Cysteine), immobilized thiol on silica (Si-Thiol), or a combination thereof.
  • an immobilized reducing agent such as immobilized diphenylphosphine on silica (Si-DPP), immobilized thiol on agarose (Ag-Thiol), immobilized cysteine on silica (Si-Cysteine), immobilized thiol on silica (Si-Thiol), or a combination thereof.
  • a reducing agent may comprise a free reducing agent, such as potassium metabisulfite, sodium thioglycolate, tris(2- carboxyethyl)phosphine (TCEP), sodium thiosulfate, N-acetyl cysteine, glutathione, dithiothreitol (DTT), cystamine, dithioerythritol (DTE), dichlorodiphenyltrichloroethane (DDT), homocysteine, lipoic acid, or a combination thereof.
  • the pH may be, or adjusted to be, a pH of from about 7 to about 9.
  • a buffer may be selected from sodium phosphate, sodium citrate, sodium succinate, histidine, histidine-HCl, sodium malate, sodium carbonate, and TRIS (tris(hydroxymethyl)aminomethane).
  • a buffer may be TRIS and may be, or adjusted to be, from about 20 mM to about 150 mM TRIS.
  • the temperature of the composition may be, or adjusted to be, 25 0C or less.
  • the composition may also comprise a free reducing agent or antioxidant. 22. Other Delivery Agents a.
  • compositions or formulations of the present disclosure comprise a delivery agent, e.g., a liposome, a lipolexes, a lipid nanoparticle, or any combination thereof.
  • a delivery agent e.g., a liposome, a lipolexes, a lipid nanoparticle, or any combination thereof.
  • the polynucleotides described herein e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide
  • Liposomes, lipoplexes, or lipid nanoparticles can be used to improve the efficacy of the polynucleotides directed protein production as these formulations can increase cell transfection by the polynucleotide; and/or increase the translation of encoded protein.
  • Attorney Docket No.45817-0124WO1 / MTX959.20 The liposomes, lipoplexes, or lipid nanoparticles can also be used to increase the stability of the polynucleotides.
  • Liposomes are artificially-prepared vesicles that can primarily be composed of a lipid bilayer and can be used as a delivery vehicle for the administration of pharmaceutical formulations. Liposomes can be of different sizes.
  • a multilamellar vesicle can be hundreds of nanometers in diameter, and can contain a series of concentric bilayers separated by narrow aqueous compartments.
  • a small unicellular vesicle SUV
  • a large unilamellar vesicle LUV
  • Liposome design can include, but is not limited to, opsonins or ligands to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis.
  • Liposomes can contain a low or a high pH value in order to improve the delivery of the pharmaceutical formulations.
  • liposomes can depend on the pharmaceutical formulation entrapped and the liposomal ingredients, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and/or delivery of the vesicles, the optimal size, polydispersity and the shelf-life of the vesicles for the intended application, and the batch-to-batch reproducibility and scale up production of safe and efficient liposomal products, etc.
  • liposomes such as synthetic membrane vesicles can be prepared by the methods, apparatus and devices described in U.S. Pub. Nos.
  • the polynucleotides described herein can be encapsulated by the liposome and/or it can be contained in an aqueous core that can then be encapsulated by the liposome as described in, e.g., Intl. Pub. Nos. WO2012031046, WO2012031043, WO2012030901, WO2012006378, and WO2013086526; and U.S. Pub. Nos.
  • the polynucleotides described herein can be formulated in a cationic oil-in-water emulsion where the emulsion particle comprises an oil core and a cationic lipid that can interact with the polynucleotide anchoring the molecule to the emulsion particle.
  • the polynucleotides described herein can be formulated in a water-in-oil emulsion comprising a continuous hydrophobic phase in which the hydrophilic phase is dispersed.
  • Exemplary emulsions can be made by the methods described in Intl. Pub. Nos. WO2012006380 and WO201087791, each of which is herein incorporated by reference in its entirety.
  • the polynucleotides described herein can be formulated in a lipid-polycation complex. The formation of the lipid-polycation complex can be accomplished by methods as described in, e.g., U.S. Pub. No. US20120178702.
  • the polycation can include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and/or polyarginine and the cationic peptides described in Intl. Pub. No. WO2012013326 or U.S. Pub. No. US20130142818.
  • a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and/or polyarginine and the cationic peptides described in Intl. Pub. No. WO2012013326 or U.S. Pub. No. US20130142818.
  • LNP lipid nanoparticle
  • Lipid nanoparticle formulations typically comprise one or more lipids.
  • the lipid is an ionizable amino lipid, sometimes referred to in the art as an “ionizable cationic lipid”.
  • lipid nanoparticle formulations further comprise other components, including a phospholipid, a structural lipid, and a molecule capable of reducing particle aggregation, for example a PEG or PEG-modified lipid.
  • Exemplary ionizable amino lipids include, but not limited to, any Compounds II, VI, A, and B disclosed herein, DLin-MC3-DMA (MC3), DLin-DMA, DLenDMA, DLin-D-DMA, DLin-K-DMA, DLin-M-C2-DMA, DLin-K-DMA, DLin- KC2-DMA, DLin-KC3-DMA, DLin-KC4-DMA, DLin-C2K-DMA, DLin-MP-DMA, Attorney Docket No.45817-0124WO1 / MTX959.20 DODMA, 98N12-5, C12-200, DLin-C-DAP, DLin-DAC, DLinDAP, DLinAP, DLin- EG-DMA, DLin-2-DMAP, KL10, KL22, KL25, Octyl-CLinDMA, Oct
  • exemplary ionizable amino lipids include, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien- 1-amine (L608), (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)- N,N-dimemylhexacosa-17,20-dien-9-amine, (16Z,19Z)-N5N-dimethylpentacosa- 16,19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16-dien-5-amine, (12Z,15Z)- N,N-dimethylhenicosa-12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17- dien-6-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-die
  • Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.
  • the Attorney Docket No.45817-0124WO1 / MTX959.20 phospholipids are DLPC, DMPC, DOPC, DPPC, DSPC, DUPC, 18:0 Diether PC, DLnPC, DAPC, DHAPC, DOPE, 4ME 16:0 PE, DSPE, DLPE,DLnPE, DAPE, DHAPE, DOPG, and any combination thereof.
  • the phospholipids are MPPC, MSPC, PMPC, PSPC, SMPC, SPPC, DHAPE, DOPG, and any combination thereof.
  • the amount of phospholipids (e.g., DSPC) in the lipid composition ranges from about 1 mol% to about 20 mol%. In some embodiments, the amount of phospholipids (e.g., DSPC) in the lipid composition ranges from about 5-15 mol%, optionally 10-12 mol%, for example, 5-6 mol%, 6-7 mol%, 7-8 mol%, 8-9 mol%, 9-10 mol%, 10-11 mol%, 11-12 mol%, 12-13 mol%, 13-14 mol%, or 14-15 mol%.
  • the structural lipids include sterols and lipids containing sterol moieties.
  • the structural lipids include cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof.
  • the structural lipid is cholesterol.
  • the amount of the structural lipids (e.g., cholesterol) in the lipid composition ranges from about 20 mol% to about 60 mol%.
  • the amount of the structural lipids (e.g., cholesterol) in the lipid composition ranges from about 30-45 mol%, optionally 35-40 mol%, for example, 30-31 mol%, 31-32 mol%, 32-33 mol%, 33-34 mol%, 35-35 mol%, 35-36 mol%, 36-37 mol%, 38-38 mol%, 38-39 mol%, or 39-40 mol%.
  • the PEG-modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2- diacyloxypropan-3-amines.
  • PEGylated lipids are also referred to as PEGylated lipids.
  • a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • the PEG-lipid are 1,2- dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG- disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG- diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG- DPPE), or PEG-l,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).
  • PEG-DMG 1,2- dimyristoyl-sn-glycerol methoxypolyethylene glycol
  • PEG-DSPE 1,2-distearoyl-sn- glycero-3-
  • the PEG moiety has a size of about 1000, 2000, 5000, 10,000, 15,000 Attorney Docket No.45817-0124WO1 / MTX959.20 or 20,000 daltons.
  • the amount of PEG-lipid in the lipid composition ranges from about 0 mol% to about 5 mol%. In some embodiments, the amount of PEG-lipid in the lipid composition ranges from about 1-5%, optionally 1-3 mol%, for example 1.5 to 2.5 mol%, 1-2 mol%, 2-3 mol%, 3-4 mol%, or 4-5 mol%.
  • the LNP formulations described herein can additionally comprise a permeability enhancer molecule.
  • Non-limiting permeability enhancer molecules are described in U.S. Pub. No. US20050222064, herein incorporated by reference in its entirety.
  • the LNP formulations can further contain a phosphate conjugate.
  • the phosphate conjugate can increase in vivo circulation times and/or increase the targeted delivery of the nanoparticle.
  • Phosphate conjugates can be made by the methods described in, e.g., Intl. Pub. No. WO2013033438 or U.S. Pub. No. US20130196948.
  • the LNP formulation can also contain a polymer conjugate (e.g., a water soluble conjugate) as described in, e.g., U.S. Pub. Nos.
  • the LNP formulations can comprise a conjugate to enhance the delivery of nanoparticles of the present invention in a subject. Further, the conjugate can inhibit phagocytic clearance of the nanoparticles in a subject.
  • the conjugate can be a "self" peptide designed from the human membrane protein CD47 (e.g., the "self” particles described by Rodriguez et al, Science 2013339, 971-975, herein incorporated by reference in its entirety). As shown by Rodriguez et al.
  • the LNP formulations can comprise a carbohydrate carrier.
  • the carbohydrate carrier can include, but is not limited to, an anhydride-modified phytoglycogen or glycogen-type material, phytoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta- dextrin (e.g., Intl. Pub. No. WO2012109121, herein incorporated by reference in its entirety).
  • the LNP formulations can be coated with a surfactant or polymer to improve the delivery of the particle.
  • the LNP can be coated Attorney Docket No.45817-0124WO1 / MTX959.20 with a hydrophilic coating such as, but not limited to, PEG coatings and/or coatings that have a neutral surface charge as described in U.S. Pub. No. US20130183244, herein incorporated by reference in its entirety.
  • the LNP formulations can be engineered to alter the surface properties of particles so that the lipid nanoparticles can penetrate the mucosal barrier as described in U.S. Pat. No.8,241,670 or Intl. Pub. No. WO2013110028, each of which is herein incorporated by reference in its entirety.
  • the LNP engineered to penetrate mucus can comprise a polymeric material (i.e., a polymeric core) and/or a polymer-vitamin conjugate and/or a tri-block co-polymer.
  • the polymeric material can include, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.
  • LNP engineered to penetrate mucus can also include surface altering agents such as, but not limited to, polynucleotides, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as for example dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g., N-acetylcysteine, mugwort, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin
  • the mucus penetrating LNP can be a hypotonic formulation comprising a mucosal penetration enhancing coating.
  • the formulation can be hypotonic for the epithelium to which it is being delivered.
  • hypotonic formulations can be found in, e.g., Intl. Pub. No. WO2013110028, herein incorporated by reference in its entirety.
  • the polynucleotide described herein is formulated as a lipoplex, such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® Attorney Docket No.45817-0124WO1 / MTX959.20 (Cambridge, MA), and polyethylenimine (PEI) or protamine-based targeted and non- targeted delivery of nucleic acids (Aleku et al. Cancer Res.200868:9788-9798; Strumberg et al.
  • a lipoplex such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® Attorney Docket No.45817-0124WO1 / MTX959.20 (Cambridge, MA), and polyethylenimine
  • the polynucleotides described herein are formulated as a solid lipid nanoparticle (SLN), which can be spherical with an average diameter between 10 to 1000 nm.
  • SLN possess a solid lipid core matrix that can solubilize lipophilic molecules and can be stabilized with surfactants and/or emulsifiers.
  • Exemplary SLN can be those as described in Intl. Pub. No. WO2013105101, herein incorporated by reference in its entirety.
  • the polynucleotides described herein can be formulated for controlled release and/or targeted delivery.
  • controlled release refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome.
  • the polynucleotides can be encapsulated into a delivery agent described herein and/or known in the art for controlled release and/or targeted delivery.
  • the term “encapsulate” means to enclose, surround or encase.
  • encapsulation can be substantial, complete or partial.
  • substantially encapsulated means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, or greater than 99% of the pharmaceutical composition or compound of the invention can be enclosed, surrounded or encased within the delivery agent.
  • Partially encapsulation means that less than 10, 10, 20, 30, 4050 or less of the pharmaceutical composition or compound of the invention can be enclosed, surrounded or encased within the delivery agent.
  • encapsulation can be determined by measuring the escape or the activity of the pharmaceutical composition or compound of the invention using fluorescence and/or electron micrograph. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, or greater than 99% of the pharmaceutical composition or compound of the invention are encapsulated in the delivery agent.
  • the polynucleotides described herein can be encapsulated in a therapeutic nanoparticle, referred to herein as "therapeutic nanoparticle polynucleotides.”
  • Therapeutic nanoparticles can be formulated by methods described in, e.g., Intl. Pub. Nos. WO2010005740, WO2010030763, WO2010005721, WO2010005723, and WO2012054923; and U.S. Pub. Nos.
  • the therapeutic nanoparticle polynucleotide can be formulated for sustained release.
  • sustained release refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time.
  • the period of time can include, but is not limited to, hours, days, weeks, months and years.
  • the sustained release nanoparticle of the polynucleotides described herein can be formulated as disclosed in Intl. Pub. No. WO2010075072 and U.S. Pub. Nos. US20100216804, US20110217377, US20120201859 and US20130150295, each of which is herein incorporated by reference in their entirety.
  • the therapeutic nanoparticle polynucleotide can be formulated to be target specific, such as those described in Intl. Pub. Nos.
  • the LNPs can be prepared using microfluidic mixers or micromixers.
  • Exemplary microfluidic mixers can include, but are not limited to, a slit interdigital Attorney Docket No.45817-0124WO1 / MTX959.20 micromixer including, but not limited to those manufactured by Microinnova (Allerheiligen bei Wildon, Austria) and/or a staggered herringbone micromixer (SHM) (see Zhigaltsevet al., "Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing," Langmuir 28:3633-40 (2012); Belliveau et al., “Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA," Molecular Therapy-Nucleic Acids.1:e37 (2012); Chen et al., “Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation," J.
  • micromixers include Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet (IJMM,) from the Institut für Mikrotechnik Mainz GmbH, Mainz Germany.
  • methods of making LNP using SHM further comprise mixing at least two input streams wherein mixing occurs by microstructure-induced chaotic advection (MICA).
  • MICA microstructure-induced chaotic advection
  • This method can also comprise a surface for fluid mixing wherein the surface changes orientations during fluid cycling.
  • Methods of generating LNPs using SHM include those disclosed in U.S. Pub. Nos. US20040262223 and US20120276209, each of which is incorporated herein by reference in their entirety.
  • the polynucleotides described herein can be formulated in lipid nanoparticles using microfluidic technology (see Whitesides, George M., "The Origins and the Future of Microfluidics," Nature 442: 368-373 (2006); and Abraham et al., "Chaotic Mixer for Microchannels," Science 295: 647- 651 (2002); each of which is herein incorporated by reference in its entirety).
  • the polynucleotides can be formulated in lipid nanoparticles using a micromixer chip such as, but not limited to, those from Harvard Apparatus (Holliston, MA) or Dolomite Microfluidics (Royston, UK).
  • a micromixer chip can be used for rapid mixing of two or more fluid streams with a split and recombine mechanism.
  • the polynucleotides described herein can be formulated in lipid nanoparticles having a diameter from about 1 nm to about 100 nm Attorney Docket No.45817-0124WO1 / MTX959.20 such as, but not limited to, about 1 nm to about 20 nm, from about 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm to about 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50
  • the lipid nanoparticles can have a diameter from about 10 to 500 nm. In one embodiment, the lipid nanoparticle can have a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm.
  • the polynucleotides can be delivered using smaller LNPs.
  • Such particles can comprise a diameter from below 0.1 ⁇ m up to 100 nm such as, but not limited to, less than 0.1 ⁇ m, less than 1.0 ⁇ m, less than 5 ⁇ m, less than 10 ⁇ m, less than 15 um, less than 20 um, less than 25 um, less than 30 um, less than 35 Attorney Docket No.45817-0124WO1 / MTX959.20 um, less than 40 um, less than 50 um, less than 55 um, less than 60 um, less than 65 um, less than 70 um, less than 75 um, less than 80 um, less than 85 um, less than 90 um, less than 95 um, less than 100 um, less than 125 um, less than 150 um, less than 175 um, less than 200 um, less than 225 um, less than 250 um, less than 275 um, less than 300 um, less than 325 um, less than 350 um, less than 375 um, less than 400 um
  • the nanoparticles and microparticles described herein can be geometrically engineered to modulate macrophage and/or the immune response.
  • the geometrically engineered particles can have varied shapes, sizes and/or surface charges to incorporate the polynucleotides described herein for targeted delivery such as, but not limited to, pulmonary delivery (see, e.g., Intl. Pub. No. WO2013082111, herein incorporated by reference in its entirety).
  • Other physical features the geometrically engineering particles can include, but are not limited to, fenestrations, angled arms, asymmetry and surface roughness, charge that can alter the interactions with cells and tissues.
  • the nanoparticles described herein are stealth nanoparticles or target-specific stealth nanoparticles such as, but not limited to, those described in U.S. Pub. No. US20130172406, herein incorporated by reference in its entirety.
  • the stealth or target-specific stealth nanoparticles can comprise a polymeric matrix, which can comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polyesters, polyanhydrides, polyethers, polyurethanes, polymethacrylates, polyacrylates, polycyanoacrylates, or combinations thereof.
  • polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyester
  • compositions or formulations of the present disclosure comprise a delivery agent, e.g., a lipidoid.
  • a delivery agent e.g., a lipidoid.
  • the polynucleotides described herein e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide
  • lipidoids Complexes, micelles, liposomes or particles can be prepared containing these lipidoids and therefore to achieve an effective delivery of the polynucleotide, as judged by the production of an encoded protein, following the injection of a lipidoid formulation via localized and/or systemic routes of administration.
  • Lipidoid complexes of polynucleotides can be administered by various means including, but not limited to, intravenous, intramuscular, or subcutaneous routes. [00708] The synthesis of lipidoids is described in literature (see Mahon et al., Bioconjug.
  • Formulations with the different lipidoids including, but not limited to penta[3-(1-laurylaminopropionyl)]-triethylenetetramine hydrochloride (TETA-5LAP; also known as 98N12-5, see Murugaiah et al., Analytical Biochemistry, 401:61 (2010)), C12-200 (including derivatives and variants), and MD1, can be tested for in vivo activity.
  • TETA-5LAP also known as 98N12-5, see Murugaiah et al., Analytical Biochemistry, 401:61 (2010)
  • C12-200 including derivatives and variants
  • MD1 penta[3-(1-laurylaminopropionyl)]-triethylenetetramine hydrochloride
  • 98N12-5LAP also known as 98N12-5, see Murugaiah et al., Analytical Biochemistry, 401:61 (2010)
  • C12-200 including derivatives and variants
  • the lipidoid "C12-200" is disclosed by Love et al., Proc Natl Acad Sci U S A.2010107:1864-1869 and Liu and Huang, Molecular Therapy.2010669-670. Each of the references is herein incorporated by reference in its entirety.
  • the polynucleotides described herein can be formulated in an aminoalcohol lipidoid.
  • Aminoalcohol lipidoids can be prepared by the methods described in U.S. Patent No.8,450,298 (herein incorporated by reference in its entirety).
  • the lipidoid formulations can include particles comprising either 3 or 4 or more components in addition to polynucleotides.
  • Lipidoids and polynucleotide formulations comprising lipidoids are described in Intl. Pub. No. WO 2015051214 (herein incorporated by reference in its entirety. Attorney Docket No.45817-0124WO1 / MTX959.20 c. Hyaluronidase [00712]
  • the polynucleotides described herein e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide
  • hyaluronidase for injection e.g., intramuscular or subcutaneous injection.
  • the polynucleotides described herein e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide
  • a nanoparticle mimic can mimic the delivery function organisms or particles such as, but not limited to, pathogens, viruses, bacteria, fungus, parasites, prions and cells.
  • the polynucleotides described herein can be encapsulated in a non-viron particle that can mimic the delivery function of a virus (see e.g., Intl. Pub. No.
  • compositions or formulations of the present disclosure comprise the polynucleotides described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide) in self-assembled nanoparticles, or amphiphilic macromolecules (AMs) for delivery.
  • polynucleotides described herein e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide
  • AMs amphiphilic macromolecules
  • AMs comprise biocompatible amphiphilic polymers that have an alkylated sugar backbone covalently linked to poly(ethylene glycol). In aqueous solution, the AMs self- assemble to form micelles. Nucleic acid self-assembled nanoparticles are described in Intl. Appl. No. PCT/US2014/027077, and AMs and methods of forming AMs are described in U.S. Pub. No. US20130217753, each of which is herein incorporated by reference in its entirety. Attorney Docket No.45817-0124WO1 / MTX959.20 f.
  • compositions or formulations of the present disclosure comprise the polynucleotides described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide) and a cation or anion, such as Z n2+ , Ca 2+ , Cu 2+ , Mg 2+ and combinations thereof.
  • exemplary formulations can include polymers and a polynucleotide complexed with a metal cation as described in, e.g., U.S. Pat. Nos.6,265,389 and 6,555,525, each of which is herein incorporated by reference in its entirety.
  • cationic nanoparticles can contain a combination of divalent and monovalent cations.
  • the delivery of polynucleotides in cationic nanoparticles or in one or more depot comprising cationic nanoparticles can improve polynucleotide bioavailability by acting as a long-acting depot and/or reducing the rate of degradation by nucleases.
  • Amino Acid Lipids [00716]
  • the compositions or formulations of the present disclosure comprise the polynucleotides described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide) that is formulation with an amino acid lipid.
  • Amino acid lipids are lipophilic compounds comprising an amino acid residue and one or more lipophilic tails.
  • Non-limiting examples of amino acid lipids and methods of making amino acid lipids are described in U.S. Pat. No. 8,501,824.
  • the amino acid lipid formulations can deliver a polynucleotide in releasable form that comprises an amino acid lipid that binds and releases the polynucleotides.
  • the release of the polynucleotides described herein can be provided by an acid-labile linker as described in, e.g., U.S. Pat.
  • compositions or formulations of the present disclosure comprise the polynucleotides described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide) in an interpolyelectrolyte complex.
  • Interpolyelectrolyte complexes are formed when charge-dynamic polymers are complexed with one or more anionic molecules.
  • compositions or formulations of the present disclosure comprise the polynucleotides described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide) in crystalline polymeric systems.
  • compositions or formulations of the present disclosure comprise the polynucleotides described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide) and a natural and/or synthetic polymer.
  • Exemplary polymers include, DYNAMIC POLYCONJUGATE® (Arrowhead Research Corp., Pasadena, CA) formulations from MIRUS® Bio (Madison, WI) and Roche Madison (Madison, WI), PHASERXTM polymer formulations such as, without limitation, SMARTT POLYMER TECHNOLOGYTM Attorney Docket No.45817-0124WO1 / MTX959.20 (PHASERX®, Seattle, WA), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, CA), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, CA), dendrimers and poly(lactic-co-glycolic acid) (PLGA) polymers.
  • PHASERXTM polymer formulations such as, without limitation, SMARTT POLYMER TECHNOLOGYTM Attorney Docket No.45817-0124WO1 / MTX959.20 (
  • the polymer formulations allow a sustained or delayed release of the polynucleotide (e.g., following intramuscular or subcutaneous injection).
  • the altered release profile for the polynucleotide can result in, for example, translation of an encoded protein over an extended period of time.
  • the polymer formulation can also be used to increase the stability of the polynucleotide.
  • Sustained release formulations can include, but are not limited to, PLGA microspheres, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, FL), HYLENEX® (Halozyme Therapeutics, San Diego CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, GA), TISSELL® (Baxter International, Inc. Deerfield, IL), PEG-based sealants, and COSEAL® (Baxter International, Inc. Deerfield, IL).
  • modified mRNA can be formulated in PLGA microspheres by preparing the PLGA microspheres with tunable release rates (e.g., days and weeks) and encapsulating the modified mRNA in the PLGA microspheres while maintaining the integrity of the modified mRNA during the encapsulation process.
  • EVAc are non-biodegradable, biocompatible polymers that are used extensively in pre-clinical sustained release implant applications (e.g., extended release products Ocusert a pilocarpine ophthalmic insert for glaucoma or progestasert a sustained release progesterone intrauterine device; transdermal delivery systems Testoderm, Duragesic and Selegiline; catheters).
  • Poloxamer F-407 NF is a hydrophilic, non-ionic surfactant triblock copolymer of polyoxyethylene- polyoxypropylene-polyoxyethylene having a low viscosity at temperatures less than 5oC and forms a solid gel at temperatures greater than 15oC.
  • the polynucleotides described herein can be formulated with the polymeric compound of PEG grafted with PLL as described in U.S. Pat. No.6,177,274.
  • the polynucleotides Attorney Docket No.45817-0124WO1 / MTX959.20 described herein can be formulated with a block copolymer such as a PLGA-PEG block copolymer (see e.g., U.S. Pub. No. US20120004293 and U.S. Pat. Nos. 8,236,330 and 8,246,968), or a PLGA-PEG-PLGA block copolymer (see e.g., U.S. Pat. No.6,004,573).
  • a block copolymer such as a PLGA-PEG block copolymer (see e.g., U.S. Pub. No. US20120004293 and U.S. Pat. Nos. 8,236,330 and 8,246,968), or a PLGA-PEG-PLGA block copolymer (see e.g., U.S. Pat. No.6,004,573).
  • a block copolymer such as
  • the polynucleotides described herein can be formulated with at least one amine-containing polymer such as, but not limited to polylysine, polyethylene imine, poly(amidoamine) dendrimers, poly(amine-co-esters) or combinations thereof.
  • amine-containing polymer such as, but not limited to polylysine, polyethylene imine, poly(amidoamine) dendrimers, poly(amine-co-esters) or combinations thereof.
  • Exemplary polyamine polymers and their use as delivery agents are described in, e.g., U.S. Pat. Nos.8,460,696, 8,236,280, each of which is herein incorporated by reference in its entirety.
  • the polynucleotides described herein can be formulated in a biodegradable cationic lipopolymer, a biodegradable polymer, or a biodegradable copolymer, a biodegradable polyester copolymer, a biodegradable polyester polymer, a linear biodegradable copolymer, PAGA, a biodegradable cross- linked cationic multi-block copolymer or combinations thereof as described in, e.g., U.S. Pat. Nos.6,696,038, 6,517,869, 6,267,987, 6,217,912, 6,652,886, 8,057,821, and 8,444,992; U.S. Pub. Nos.
  • the polynucleotides described herein can be formulated in or with at least one cyclodextrin polymer as described in U.S. Pub. No. US20130184453. In some embodiments, the polynucleotides described herein can be formulated in or with at least one crosslinked cation-binding polymers as described in Intl. Pub. Nos.
  • the polynucleotides described herein can be formulated in or with at least PEGylated albumin polymer as described in U.S. Pub. No. US20130231287. Each of the references is herein incorporated by reference in its entirety. [00727] In some embodiments, the polynucleotides disclosed herein can be formulated as a nanoparticle using a combination of polymers, lipids, and/or other biodegradable agents, such as, but not limited to, calcium phosphate.
  • Components can be combined in a core-shell, hybrid, and/or layer-by-layer architecture, to allow for Attorney Docket No.45817-0124WO1 / MTX959.20 fine-tuning of the nanoparticle for delivery (Wang et al., Nat Mater.20065:791-796; Fuller et al., Biomaterials.200829:1526-1532; DeKoker et al., Adv Drug Deliv Rev. 201163:748-761; Endres et al., Biomaterials.201132:7721-7731; Su et al., Mol Pharm.2011 Jun 6;8(3):774-87; herein incorporated by reference in their entireties).
  • the nanoparticle can comprise a plurality of polymers such as, but not limited to hydrophilic-hydrophobic polymers (e.g., PEG-PLGA), hydrophobic polymers (e.g., PEG) and/or hydrophilic polymers (Intl. Pub. No. WO20120225129, herein incorporated by reference in its entirety).
  • hydrophilic-hydrophobic polymers e.g., PEG-PLGA
  • hydrophobic polymers e.g., PEG
  • hydrophilic polymers e.g., PEG-PLGA
  • hydrophobic polymers e.g., PEG
  • hydrophilic polymers e.g., PEG
  • hydrophilic polymers e.g., PEG-PLGA
  • hydrophobic polymers e.g., PEG
  • hydrophilic polymers e.g., PEG-PLGA
  • hydrophobic polymers e.g., PEG
  • hydrophilic polymers e.g
  • the complexation, delivery, and internalization of the polymeric nanoparticles can be precisely controlled by altering the chemical composition in both the core and shell components of the nanoparticle.
  • the core-shell nanoparticles can efficiently deliver siRNA to mouse hepatocytes after they covalently attach cholesterol to the nanoparticle.
  • a hollow lipid core comprising a middle PLGA layer and an outer neutral lipid layer containing PEG can be used to delivery of the polynucleotides as described herein.
  • the lipid nanoparticles can comprise a core of the polynucleotides disclosed herein and a polymer shell, which is used to protect the polynucleotides in the core.
  • the polymer shell can be any of the polymers described herein and are known in the art.
  • the polymer shell can be used to protect the polynucleotides in the core.
  • Core–shell nanoparticles for use with the polynucleotides described herein are described in U.S. Pat. No.8,313,777 or Intl. Pub. No. WO2013124867, each of which is herein incorporated by reference in their entirety. k.
  • compositions or formulations of the present disclosure comprise the polynucleotides described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide) that is formulated with peptides and/or proteins to increase transfection of cells by the polynucleotide, and/or to alter the biodistribution of the polynucleotide (e.g., by targeting specific Attorney Docket No.45817-0124WO1 / MTX959.20 tissues or cell types), and/or increase the translation of encoded protein (e.g., Intl. Pub. Nos.
  • the peptides can be those described in U.S. Pub. Nos. US20130129726, US20130137644 and US20130164219. Each of the references is herein incorporated by reference in its entirety. l.
  • compositions or formulations of the present disclosure comprise the polynucleotides described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Snu13 polypeptide) that is covalently linked to a carrier or targeting group, or including two encoding regions that together produce a fusion protein (e.g., bearing a targeting group and therapeutic protein or peptide) as a conjugate.
  • the conjugate can be a peptide that selectively directs the nanoparticle to neurons in a tissue or organism, or assists in crossing the blood-brain barrier.
  • the conjugates include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); an carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid.
  • the ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid, an oligonucleotide (e.g., an aptamer).
  • polyamino acids examples include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2- hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N- isopropylacrylamide polymers, or polyphosphazine.
  • PLL polylysine
  • poly L-aspartic acid poly L-glutamic acid
  • styrene-maleic acid anhydride copolymer poly(L-lactide-co-glycolied) copolymer
  • divinyl ether-maleic anhydride copolymer divinyl ether
  • polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
  • the conjugate can function as a carrier for the polynucleotide disclosed herein.
  • the conjugate can comprise a cationic polymer such Attorney Docket No.45817-0124WO1 / MTX959.20 as, but not limited to, polyamine, polylysine, polyalkylenimine, and polyethylenimine that can be grafted to with poly(ethylene glycol).
  • a cationic polymer such as Attorney Docket No.45817-0124WO1 / MTX959.20 as, but not limited to, polyamine, polylysine, polyalkylenimine, and polyethylenimine that can be grafted to with poly(ethylene glycol).
  • Exemplary conjugates and their preparations are described in U.S. Pat. No.6,586,524 and U.S. Pub. No. US20130211249, each of which herein is incorporated by reference in its entirety.
  • the conjugates can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell.
  • a cell or tissue targeting agent e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell.
  • a targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N- acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGD peptide mimetic or an aptamer.
  • Targeting groups can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as an endothelial cell or bone cell.
  • Targeting groups can also include hormones and hormone receptors. They can also include non- peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl- glucosamine multivalent mannose, multivalent frucose, or aptamers.
  • the ligand can be, for example, a lipopolysaccharide, or an activator of p38 MAP kinase.
  • the targeting group can be any ligand that is capable of targeting a specific receptor. Examples include, without limitation, folate, GalNAc, galactose, mannose, mannose-6P, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands.
  • the targeting group is an aptamer.
  • the aptamer can be unmodified or have any combination of modifications disclosed herein.
  • the targeting group can be a glutathione receptor (GR)-binding conjugate for targeted delivery across the blood- central nervous system barrier as described in, e.g., U.S. Pub. No. US2013021661012 (herein incorporated by reference in its entirety).
  • the conjugate can be a synergistic biomolecule- polymer conjugate, which comprises a long-acting continuous-release system to provide a greater therapeutic efficacy.
  • the synergistic biomolecule-polymer conjugate can be those described in U.S. Pub. No.
  • the conjugate can be an aptamer conjugate as described in Intl. Pat. Pub. No. WO2012040524.
  • the conjugate can be an amine containing polymer conjugate as described in U.S. Pat. No.8,507,653.
  • the polynucleotides can be conjugated to SMARTT POLYMER TECHNOLOGY® (PHASERX®, Inc. Seattle, WA).
  • the polynucleotides described herein are covalently conjugated to a cell penetrating polypeptide, which can also include a signal sequence or a targeting sequence.
  • the conjugates can be designed to have increased stability, and/or increased cell transfection; and/or altered the biodistribution (e.g., targeted to specific tissues or cell types).
  • the polynucleotides described herein can be conjugated to an agent to enhance delivery.
  • the agent can be a monomer or polymer such as a targeting monomer or a polymer having targeting blocks as described in Intl. Pub. No. WO2011062965.
  • the agent can be a transport agent covalently coupled to a polynucleotide as described in, e.g., U.S. Pat. Nos.6,835.393 and 7,374,778.
  • the agent can be a membrane barrier transport enhancing agent such as those described in U.S. Pat. Nos.7,737,108 and 8,003,129. Each of the references is herein incorporated by reference in its entirety. 23. Methods of Use [00741]
  • Snu13 polynucleotides, pharmaceutical compositions and formulations described above are used in the preparation, manufacture and therapeutic use of compositions to express Snu13 and/or to regulate expression of a polypeptide.
  • the Snu13 polynucleotides, compositions and formulations of the present disclosure are used to regulate expression of a polypeptide (e.g., a target Attorney Docket No.45817-0124WO1 / MTX959.20 polypeptide), wherein the polypeptide is encoded by a polynucleotide comprising a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500) and an ORF encoding the polypeptide.
  • a polypeptide e.g., a target Attorney Docket No.45817-0124WO1 / MTX959.20 polypeptide
  • a polypeptide e.g., a target Attorney Docket No.45817-0124WO1 / MTX959.20 polypeptide
  • the polypeptide is encoded by a polynucleotide comprising a Snu
  • the Snu13 polynucleotides are used to preferentially regulate expression of a polypeptide encoded by a G5 polynucleotide (compared to an unmodified polynucleotide).
  • a method for regulating expression of a polypeptide in a subject comprising administering to the subject: (i) a first polynucleotide comprising (a) a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500), and (b) an open reading frame encoding a first polypeptide; and (ii) a second polynucleotide comprising a nucleotide sequence encoding a second polypeptide, wherein the second polypeptide comprises a Snu13 polypeptide described herein.
  • a first polynucleotide comprising (a) a Snu13-binding site (e.g.
  • binding of the second polypeptide to the Snu13-binding site represses translation of the first polypeptide from the first polynucleotide.
  • translation of the first polypeptide is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at lease 90%, at least 95%, or 100% (compared to translation of the first polypeptide in the absence of the second polynucleotide).
  • a method for regulating expression of a polypeptide comprising contacting a cell with: (i) a first polynucleotide comprising (a) a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500), and (b) an open reading frame encoding a first polypeptide; and (ii) a second polynucleotide comprising a nucleotide sequence encoding a second polypeptide, wherein the second polypeptide comprises a Snu13 polypeptide described herein.
  • a Snu13-binding site e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500
  • an open reading frame encoding a first polypeptide
  • a second polynucleotide comprising a nucleotide sequence encoding a second
  • binding of the second polypeptide to the Snu13-binding site represses translation of the first polypeptide from the first polynucleotide.
  • translation of the first polypeptide is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at lease 90%, at least 95%, or 100% (compared to translation of the first polypeptide in the absence of the second polynucleotide).
  • the polynucleotides, pharmaceutical compositions and formulations of the invention are used in methods for increasing the level of Snu13 in a subject in need thereof.
  • the polynucleotides, pharmaceutical compositions and formulations of the invention are used in methods for increasing the level of a target polypeptide in a subject in thereof (e.g., by administering to the subject in need thereof a Snu13 polynucleotide described herein (or a composition or formulation comprising same) and a polynucleotide comprising a Snu13-binding site and an ORF encoding the target polypeptide).
  • the administration of a composition or formulation comprising polynucleotide encoding Snu13 of the present disclosure to a subject results in an increase in Snu13 protein in cells to a level at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or to 100% higher than the level observed prior to the administration of the composition or formulation.
  • the administration of a composition or formulation comprising polynucleotide encoding Snu13 of the present disclosure to a subject results in an increase in a target polypeptide in cells to a level at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or to 100% higher than the level observed prior to the administration of the composition or formulation.
  • the administration of the polynucleotide encoding Snu13, pharmaceutical composition or formulation of the present disclosure results in expression of Snu13 protein in cells of the subject.
  • administering the polynucleotide encoding Snu13, pharmaceutical composition or formulation of the present disclosure results in an increase of Snu13 protein activity in the subject.
  • the polynucleotides of the present disclosure are used in methods of administering a composition or formulation comprising an mRNA encoding a Snu13 polypeptide to a subject, wherein the method results in an increase of Snu13 protein activity in at least some cells of a subject.
  • the administration of the polynucleotide encoding Snu13, pharmaceutical composition or formulation of the present disclosure results in expression of Snu13 protein and a target polypeptide in cells of the subject.
  • administering the polynucleotide encoding Snu13, pharmaceutical composition or formulation of the present disclosure results in an increase of Snu13 protein activity and of a target polypeptide in the subject.
  • the polynucleotides of the present disclosure are used in methods of administering a composition or formulation comprising an mRNA encoding a Snu13 polypeptide to a subject, in combination with a polynucleotide comprising a Snu13- binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500) and an ORF encoding a target polypeptide, wherein the method results in an increase of Snu13 protein activity and an increase in the target polypeptide activity in at least some cells of a subject.
  • a polynucleotide comprising a Snu13- binding site e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500
  • an ORF encoding a target polypeptide
  • the administration of a composition or formulation comprising an mRNA encoding a Snu13 polypeptide to a subject results in an increase of Snu13 protein activity in cells subject to a level at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or to 100% or more of the activity level expected in a normal subject, e.g., a human not suffering from deficiency in Snu13 or defective Snu13.
  • Snu13 activity can be evaluated using assays known in the art and as described herein.
  • the polynucleotides encoding Snu13, pharmaceutical compositions, or formulations of the present disclosure can be repeatedly administered such that Snu13 protein, respectively, is expressed at a therapeutic level for a period of time sufficient to have a beneficial biological effect as described herein (e.g., regulation of a target polypeptide encoded by a polynucleotide comprising a Snu13-binding site).
  • the method or use comprises administering a polynucleotide, e.g., mRNA, comprising a nucleotide sequence having sequence similarity (e.g., at least 65% identity) to a polynucleotide of any one of SEQ ID Attorney Docket No.45817-0124WO1 / MTX959.20 NOs:300-317, wherein the polynucleotide encodes a Snu13 polypeptide described herein (e.g., any one of SEQ ID NOs: 320-373).
  • Other aspects of the present disclosure relate to transplantation of cells containing polynucleotides to a mammalian subject.
  • the present disclosure also provides methods to increase Snu13 activity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition or formulation comprising mRNA encoding a Snu13 polypeptide disclosed herein.
  • the method further comprises administering to the subject in need thereof a therapeutically effective amount of a composition or formulation comprising an mRNA encoding a target polypeptide, wherein the mRNA encoding the target polypeptide comprises a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500).
  • the mRNA encoding the target polypeptide is G5 modified.
  • the subject in need thereof is in need of the target polypeptide (e.g., is deficient in the target polypeptide or has a defective target polypeptide).
  • the polynucleotides e.g., mRNA
  • pharmaceutical compositions and formulations used in the methods of the invention comprise a uracil-modified sequence encoding a Snu13 polypeptide disclosed herein and a miRNA binding site disclosed herein, e.g., a miRNA binding site that binds to miR- 142 and/or a miRNA binding site that binds to miR-126.
  • the uracil-modified sequence encoding a Snu13 polypeptide comprises at least one chemically modified nucleobase, e.g., N1-methylpseudouracil or 5-methoxyuracil.
  • At least 95% of a type of nucleobase (e.g., uracil) in a uracil- modified sequence encoding a Snu13 polypeptide of the invention are modified nucleobases.
  • at least 95% of uracil in a uracil-modified sequence encoding a Snu13 polypeptide is 1-N-methylpseudouridine or 5- methoxyuridine.
  • the polynucleotide e.g., a RNA, e.g., an mRNA
  • a delivery agent comprising, e.g., a Attorney Docket No.45817-0124WO1 / MTX959.20 compound having the Formula (I), e.g., Compound II or Compound B; or a compound having the Formula (III), (IV), (V), or (VI), e.g., Compound I or Compound VI, or any combination thereof.
  • the delivery agent comprises an ionizable amino lipid (e.g., Compound II, VI, or B), a helper lipid (e.g., DSPC), a sterol (e.g., Cholesterol), and a PEG lipid (e.g., Compound I or PEG-DMG), e.g., with a mole ratio in the range of about (i) 40-50 mol% ionizable amino lipid (e.g., Compound II, VI, or B), optionally 45-50 mol% ionizable amino lipid, for example, 45-46 mol%, 46-47 mol%, 47-48 mol%, 48-49 mol%, or 49-50 mol% for example about 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol%, 48.5 mol%, 49 mol%, or 49.5 mol%; (ii) 30-45
  • the delivery agent comprises Compound II, Cholesterol, DSPC, and Compound I.
  • the therapeutic effectiveness of a drug or a treatment of the instant invention can be characterized or determined by measuring the level of expression of an encoded protein in a sample or in samples taken from a subject (e.g., from a preclinical test subject (rodent, primate, etc.) or from a clinical subject (human)).
  • the therapeutic effectiveness of a drug or a treatment of the instant invention can be characterized or determined by measuring the level of activity of an encoded protein in a sample or in samples taken from a subject (e.g., from a preclinical test subject (rodent, primate, etc.) or from a clinical subject (human).
  • a subject e.g., from a preclinical test subject (rodent, primate, etc.) or from a clinical subject (human).
  • the therapeutic effectiveness of a drug or a treatment of the instant invention can be characterized or determined by measuring the level of an appropriate biomarker in sample(s) taken from a subject.
  • Protein Expression Levels can be determined post-administration with a single dose of an mRNA therapeutic of the invention or can be determined and/or monitored at several time Attorney Docket No.45817-0124WO1 / MTX959.20 points following administration with a single dose or can be determined and/or monitored throughout a course of treatment, e.g., a multi-dose treatment.
  • Protein Expression Levels [00757] Certain aspects of the invention feature measurement, determination and/or monitoring of the expression level or levels of protein (e.g., Snu13 or a target polypeptide) in a subject, for example, in an animal (e.g., rodents, primates, and the like) or in a human subject.
  • Animals include normal, healthy or wildtype animals, as well as animal disease models.
  • Snu13 protein or target polypeptide expression levels can be measured or determined by any art-recognized method for determining protein levels in biological samples, e.g., blood or bone marrow cells.
  • level or “level of a protein” as used herein, preferably means the weight, mass or concentration of the protein within a sample or a subject. It will be understood by the skilled artisan that in certain embodiments the sample may be subjected, e.g., to any of the following: purification, precipitation, separation, e.g.
  • compositions and Formulations for Use Certain aspects of the invention are directed to compositions or formulations comprising any of the polynucleotides disclosed above. In some instances, the composition or formulation comprises a polynucleotide encoding a Snu13 polypeptide.
  • the composition or formulation comprises a first polynucleotide encoding a Snu13 polypeptide and a second polynucleotide comprising a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500) and an ORF encoding a second polypeptide.
  • a Snu13-binding site e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500
  • the composition or formulation comprises: (i) a polynucleotide (e.g., a RNA, e.g., an mRNA) comprising a sequence- optimized nucleotide sequence (e.g., an ORF) encoding a Snu13 polypeptide, wherein Attorney Docket No.45817-0124WO1 / MTX959.20 the polynucleotide comprises at least one chemically modified nucleobase, e.g., N1-methylpseudouracil or 5-methoxyuracil (e.g., wherein at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the uracils are N1-methylpseudouracils or 5-methoxyuracils), and wherein the polynucleo
  • the delivery agent is a lipid nanoparticle comprising Compound II, Compound VI, a salt or a stereoisomer thereof, or any combination thereof.
  • the delivery agent comprises an ionizable amino lipid (e.g., Compound II, VI, or B), a helper lipid (e.g., DSPC), a sterol (e.g., Cholesterol), and a PEG lipid (e.g., Compound I or PEG-DMG), e.g., with a mole ratio in the range of about (i) 40- 50 mol% ionizable amino lipid (e.g., Compound II, VI, or B), optionally 45-50 mol% ionizable amino lipid, for example, 45-46 mol%, 46-47 mol%, 47-48 mol%, 48-49 mol%, or 49-50 mol% for example about 45 mol%, 45.5 mol%, 46 mol%, 46.5
  • the delivery agent comprises Compound II, Cholesterol, DSPC, and Compound I. Attorney Docket No.45817-0124WO1 / MTX959.20
  • the uracil or thymine content of the ORF relative to the theoretical minimum uracil or thymine content of a nucleotide sequence encoding the Snu13 polypeptide is between about 100% and about 150%.
  • the polynucleotides, compositions or formulations above are used to express a Snu13 polypeptide in a subject.
  • the polynucleotides, compositions or formulations above are used to regulate expression of a polypeptide (e.g., a polypeptide encoded by a polynucleotide comprising a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500)).
  • a polypeptide e.g., a polypeptide encoded by a polynucleotide comprising a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., SEQ ID NO:500)
  • a polypeptide e.g., a polypeptide encoded by a polynucleotide comprising a Snu13-binding site (e.g., a sequence set forth in any one of SEQ ID NOs:500-521, e.g., S
  • enteral into the intestine
  • gastroenteral gastroenteral
  • epidural into the dura matter
  • oral by way of the mouth
  • transdermal peridural
  • intracerebral into the cerebrum
  • intracerebroventricular into the cerebral ventricles
  • epicutaneous application onto the skin
  • intradermal into the skin itself
  • subcutaneous under the skin
  • nasal administration through the nose
  • intravenous bolus intravenous drip
  • intraarterial into an artery
  • intramuscular intramuscular
  • intracardiac into the heart
  • intraosseous infusion into the bone marrow
  • intrathecal into the spinal canal
  • intraperitoneal infusion or injection into the peritoneum
  • intravesical infusion intravitreal, (through the eye), intracavernous injection (into a pathologic cavity) intracavitary (into the base of the penis), intravaginal administration, intrauterine, extra-amni
  • compositions can be administered in a way that allows them cross the blood-brain barrier, vascular barrier, or other epithelial barrier.
  • a formulation for a route of administration can include at least one inactive ingredient. 26.
  • the invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
  • the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.
  • the terms “a” (or “an”), as well as the terms “one or more,” and “at least one” can be used interchangeably herein.
  • the term “a” or “an” means “single.”
  • the term “a” or “an” includes “two or more” or “multiple.”
  • the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone).
  • the term “and/or” as used in a phrase such as "A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • Nucleobases are referred to herein by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Accordingly, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, U represents uracil. Attorney Docket No.45817-0124WO1 / MTX959.20 [00772] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation.
  • Administered in combination means that two or more agents are administered to a subject at the same time or within an interval such that there can be an overlap of an effect of each agent on the patient. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minute of one another. In some embodiments, the administrations of the agents are spaced sufficiently closely together such that a combinatorial (e.g., a synergistic) effect is achieved.
  • a combinatorial e.g., a synergistic
  • amino acid substitution refers to replacing an amino acid residue present in a parent or reference sequence (e.g., a wild type Snu13 sequence) with another amino acid residue.
  • An amino acid can be substituted in a parent or reference sequence (e.g., a wild type Snu13 sequence), for example, via chemical peptide synthesis or through recombinant methods known in the art. Accordingly, a reference to a "substitution at position X" refers to the substitution of an amino acid present at position X with an alternative amino acid residue.
  • substitution patterns can be described according to the schema AnY, wherein A is the single letter code corresponding to the amino acid naturally or originally present at position n, and Y is the substituting amino acid residue.
  • substitution patterns can be described according to the schema An(YZ), wherein A is the single letter code corresponding to the amino acid Attorney Docket No.45817-0124WO1 / MTX959.20 residue substituting the amino acid naturally or originally present at position X, and Y and Z are alternative substituting amino acid residue.
  • substitutions are conducted at the nucleic acid level, i.e., substituting an amino acid residue with an alternative amino acid residue is conducted by substituting the codon encoding the first amino acid with a codon encoding the second amino acid.
  • Animal refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans at any stage of development. In some embodiments, “animal” refers to non-human animals at any stage of development.
  • the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig).
  • animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms.
  • the animal is a transgenic animal, genetically-engineered animal, or a clone.
  • the term “approximately” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • the term “associated with” means that the symptom, measurement, characteristic, or status in question is linked to the diagnosis, development, presence, or progression of that disease.
  • association can, but need not, be causatively linked to the disease.
  • association When used with respect to two or more moieties, the terms “associated with,” “conjugated,” “linked,” “attached,” and “tethered,” when used with respect to two or more moieties, means that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., Attorney Docket No.45817-0124WO1 / MTX959.20 physiological conditions.
  • An “association” need not be strictly through direct covalent chemical bonding.
  • Biocompatible As used herein, the term “biocompatible” means compatible with living cells, tissues, organs or systems posing little to no risk of injury, toxicity or rejection by the immune system.
  • Biodegradable As used herein, the term “biodegradable” means capable of being broken down into innocuous products by the action of living things.
  • Biologically active As used herein, the phrase “biologically active” refers to a characteristic of any substance that has activity in a biological system and/or organism.
  • a substance that, when administered to an organism, has a biological effect on that organism is considered to be biologically active.
  • a polynucleotide of the present invention can be considered biologically active if even a portion of the polynucleotide is biologically active or mimics an activity considered biologically relevant.
  • Chimera As used herein, "chimera” is an entity having two or more incongruous or heterogeneous parts or regions.
  • a chimeric molecule can comprise a first part comprising a Snu13 polypeptide, and a second part (e.g., genetically fused to the first part) comprising a second therapeutic protein (e.g., a protein with a distinct enzymatic activity, an antigen binding moiety, or a moiety capable of extending the plasma half life of Snu13, for example, an Fc region of an antibody).
  • a second therapeutic protein e.g., a protein with a distinct enzymatic activity, an antigen binding moiety, or a moiety capable of extending the plasma half life of Snu13, for example, an Fc region of an antibody.
  • Sequence optimization refers to a process or series of processes by which nucleobases in a reference nucleic acid sequence are replaced with alternative nucleobases, resulting in a nucleic acid sequence with improved properties, e.g., improved protein expression or decreased immunogenicity.
  • sequence optimization In general, the goal in sequence optimization is to produce a synonymous nucleotide sequence than encodes the same polypeptide sequence encoded by the reference nucleotide sequence.
  • Codon substitution refers to replacing a codon present in a reference nucleic acid sequence with another codon.
  • a codon can be substituted in a reference nucleic acid sequence, for example, via chemical peptide synthesis or through recombinant methods known in the art.
  • references to a "substitution” or “replacement” at a certain location in a nucleic acid sequence (e.g., an mRNA) or within a certain region or subsequence of a nucleic acid sequence (e.g., an mRNA) refer to the substitution of a codon at such location or region with an alternative codon.
  • the terms "coding region” and “region encoding” and grammatical variants thereof, refer to an Open Reading Frame (ORF) in a polynucleotide that upon expression yields a polypeptide or protein.
  • ORF Open Reading Frame
  • stereoisomer means any geometric isomer (e.g., cis- and trans- isomer), enantiomer, or diastereomer of a compound.
  • stereomerically pure forms e.g., geometrically pure, enantiomerically pure, or diastereomerically pure
  • enantiomeric and stereoisomeric mixtures e.g., racemates.
  • isotopes refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei.
  • isotopes of hydrogen include tritium and deuterium.
  • a compound, salt, or complex of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.
  • contacting a mammalian cell with a nanoparticle composition means that the mammalian cell and a nanoparticle are made to share a physical connection.
  • Methods of contacting cells Attorney Docket No.45817-0124WO1 / MTX959.20 with external entities both in vivo and ex vivo are well known in the biological arts.
  • contacting a nanoparticle composition and a mammalian cell disposed within a mammal can be performed by varied routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and can involve varied amounts of nanoparticle compositions.
  • routes of administration e.g., intravenous, intramuscular, intradermal, and subcutaneous
  • more than one mammalian cell can be contacted by a nanoparticle composition.
  • a "conservative amino acid substitution” is one in which the amino acid residue in a protein sequence is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, try
  • amino acid substitution is considered to be conservative.
  • a string of amino acids can be conservatively replaced with a structurally similar string that differs in order and/or composition of side chain family members.
  • Non-conservative amino acid substitutions include those in which (i) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, Ile, Phe or Val), (iii) a cysteine or proline is substituted for, or by, any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly).
  • an electropositive side chain e.g., Arg, His or Lys
  • an electronegative residue e.g., Glu or As
  • amino acid substitutions can be readily identified by workers of ordinary skill.
  • a substitution can be taken from any one of D-alanine, glycine, beta-alanine, L-cysteine and D-cysteine.
  • a replacement can be any one of D-lysine, arginine, D-arginine, homo- Attorney Docket No.45817-0124WO1 / MTX959.20 arginine, methionine, D-methionine, ornithine, or D- ornithine.
  • substitutions in functionally important regions that can be expected to induce changes in the properties of isolated polypeptides are those in which (i) a polar residue, e.g., serine or threonine, is substituted for (or by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, or alanine; (ii) a cysteine residue is substituted for (or by) any other residue; (iii) a residue having an electropositive side chain, e.g., lysine, arginine or histidine, is substituted for (or by) a residue having an electronegative side chain, e.g., glutamic acid or aspartic acid; or (iv) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having such a side chain, e.g., glycine.
  • a polar residue e.g
  • conserved refers to nucleotides or amino acid residues of a polynucleotide sequence or polypeptide sequence, respectively, that are those that occur unaltered in the same position of two or more sequences being compared. Nucleotides or amino acids that are relatively conserved are those that are conserved amongst more related sequences than nucleotides or amino acids appearing elsewhere in the sequences.
  • two or more sequences are said to be “completely conserved” if they are 100% identical to one another. In some embodiments, two or more sequences are said to be "highly conserved” if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another. In some embodiments, two or more sequences are said to be "highly conserved” if they are about 70% identical, about 80% identical, about 90% identical, about 95%, about 98%, or about 99% identical to one another.
  • two or more sequences are said to be "conserved” if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another. In some embodiments, two or more sequences are said to be "conserved” if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about Attorney Docket No.45817-0124WO1 / MTX959.20 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to one another.
  • Controlled Release refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome.
  • Cyclic or Cyclized refers to the presence of a continuous loop. Cyclic molecules need not be circular, only joined to form an unbroken chain of subunits. Cyclic molecules such as the engineered RNA or mRNA of the present invention can be single units or multimers or comprise one or more components of a complex or higher order structure.
  • Delivering means providing an entity to a destination.
  • delivering a polynucleotide to a subject can involve administering a nanoparticle composition including the polynucleotide to the subject (e.g., by an intravenous, intramuscular, intradermal, or subcutaneous route).
  • Administration of a nanoparticle composition to a mammal or mammalian cell can involve contacting one or more cells with the nanoparticle composition.
  • Delivery Agent refers to any substance that facilitates, at least in part, the in vivo, in vitro, or ex vivo delivery of a polynucleotide to targeted cells.
  • Domain As used herein, when referring to polypeptides, the term “domain” refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions).
  • Dosing regimen As used herein, a “dosing regimen” or a “dosing regimen” is a schedule of administration or physician determined regimen of treatment, prophylaxis, or palliative care.
  • Effective Amount As used herein, the term “effective amount” of an agent is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an "effective amount” depends upon the context in which it is being applied.
  • an effective amount of an agent is, for example, an amount of mRNA expressing sufficient protein, respectively, to ameliorate, reduce, eliminate, or prevent the symptoms associated with the protein deficiency, respectively, as compared to the severity of the symptom observed without administration of the agent.
  • the term "effective amount” can be used interchangeably with "effective dose,” “therapeutically effective amount,” or “therapeutically effective dose.”
  • Encapsulate means to enclose, surround or encase.
  • Encapsulation efficiency refers to the amount of a polynucleotide that becomes part of a nanoparticle composition, relative to the initial total amount of polynucleotide used in the preparation of a nanoparticle composition. For example, if 97 mg of polynucleotide are encapsulated in a nanoparticle composition out of a total 100 mg of polynucleotide initially provided to the composition, the encapsulation efficiency can be given as 97%. As used herein, “encapsulation” can refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement.
  • enhanced delivery means delivery of more (e.g., at least 1.5 fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold more) of a polynucleotide by a nanoparticle to a target tissue of interest (e.g., mammalian liver) compared to the level of delivery of a polynucleotide by a control nanoparticle to a target tissue of interest (e.g., MC3, KC2, or DLinDMA).
  • a target tissue of interest e.g., mammalian liver
  • the level of delivery of a nanoparticle to a particular tissue can be measured by comparing the amount of protein produced in a tissue to the weight of said tissue, comparing the amount of polynucleotide in a tissue to the weight of said tissue, comparing the amount of protein produced in a tissue to the amount of total protein in said tissue, or comparing the amount of polynucleotide in a tissue to the amount of total polynucleotide in said tissue.
  • a surrogate such as an animal model (e.g., a rat model).
  • expression refers to one or more of the following events: (1) production of an mRNA template from a DNA sequence (e.g., by transcription); (2) processing of an mRNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or 3′ end processing); (3) translation of an mRNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.
  • a "formulation” includes at least a polynucleotide and one or more of a carrier, an excipient, and a delivery agent.
  • Fragment refers to a portion.
  • fragments of proteins can comprise polypeptides obtained by digesting full-length protein isolated from cultured cells.
  • a fragment is a subsequences of a full length protein (e.g., Snu13) wherein N-terminal, and/or C- terminal, and/or internal subsequences have been deleted.
  • the fragments of a protein of the present invention are functional fragments.
  • a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
  • a functional fragment of a polynucleotide of the present invention is a polynucleotide capable of expressing a functional Snu13 fragment.
  • a functional fragment of Snu13 refers to a fragment of wild type Snu13 (i.e., a fragment of any of its naturally occurring isoforms), or a mutant or variant thereof, wherein the fragment retains a least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the biological activity of the corresponding full length protein.
  • helper lipid refers to a compound or molecule that includes a lipidic moiety (for insertion into a lipid layer, e.g., lipid bilayer) and a polar moiety (for interaction with physiologic solution at the surface of the lipid layer).
  • the helper lipid is a phospholipid.
  • a function of the helper lipid is to “complement” the amino lipid and increase the fusogenicity of the bilayer Attorney Docket No.45817-0124WO1 / MTX959.20 and/or to help facilitate endosomal escape, e.g., of nucleic acid delivered to cells.
  • homology refers to the overall relatedness between polymeric molecules, e.g. between nucleic acid molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Generally, the term “homology” implies an evolutionary relationship between two molecules. Thus, two molecules that are homologous will have a common evolutionary ancestor. In the context of the present invention, the term homology encompasses both to identity and similarity.
  • polymeric molecules are considered to be "homologous" to one another if at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the monomers in the molecule are identical (exactly the same monomer) or are similar (conservative substitutions).
  • the term “homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).
  • Identity refers to the overall monomer conservation between polymeric molecules, e.g., between polynucleotide molecules (e.g.
  • DNA molecules and/or RNA molecules DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • Calculation of the percent identity of two polynucleotide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence.
  • the nucleotides at corresponding nucleotide positions are then compared.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • Attorney Docket No.45817-0124WO1 / MTX959.20 The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent.
  • Suitable software programs are available from various sources, and for alignment of both protein and nucleotide sequences.
  • One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of program available from the U.S. government's National Center for Biotechnology Information BLAST web site (blast.ncbi.nlm.nih.gov).
  • Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm.
  • BLASTN is used to compare nucleic acid sequences
  • BLASTP is used to compare amino acid sequences.
  • percent sequence identity value is rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.
  • sequence alignments can be generated by integrating sequence data with data from heterogeneous sources such as structural data (e.g., crystallographic protein structures), functional data (e.g., location of mutations), or phylogenetic data.
  • structural data e.g., crystallographic protein structures
  • functional data e.g., location of mutations
  • phylogenetic data e.g., phylogenetic data.
  • T-Coffee available at www.tcoffee.org, and alternatively available, e.g., from the EBI.
  • Insertional and deletional variants when referring to polypeptides are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native or starting sequence. "Immediately adjacent" to an amino acid means connected to either the alpha-carboxy or alpha- amino functional group of the amino acid.
  • “Deletional variants” when referring to polypeptides are those with one or more amino acids in the native or starting amino acid sequence removed. Ordinarily, deletional variants will have one or more amino acids deleted in a particular region of the molecule.
  • Intact As used herein, in the context of a polypeptide, the term “intact” means retaining an amino acid corresponding to the wild type protein, e.g., not mutating or substituting the wild type amino acid. Conversely, in the context of a nucleic acid, the term “intact” means retaining a nucleobase corresponding to the wild type nucleic acid, e.g., not mutating or substituting the wild type nucleobase.
  • Ionizable amino lipid includes those lipids having one, two, three, or more fatty acid or fatty alkyl chains and a pH- titratable amino head group (e.g., an alkylamino or dialkylamino head group).
  • An ionizable amino lipid is typically protonated (i.e., positively charged) at a pH below the pKa of the amino head group and is substantially not charged at a pH above the pKa.
  • Such ionizable amino lipids include, but are not limited to DLin-MC3-DMA (MC3), (13Z,165Z)-N,N-dimethyl-3-nonydocosa-13-16-dien-1-amine (L608), and a compound of any one of Formula I, II, and II described herein (e.g., any one of Compound II, Compound VI, and Compound B).
  • Linker refers to a group of atoms, e.g., 10-1,000 atoms, and can be comprised of the atoms or groups such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine.
  • the linker can be attached to a modified nucleoside or nucleotide on the nucleobase or sugar moiety at a first end, and to a payload, e.g., a detectable or therapeutic agent, at a second end.
  • the linker can be of sufficient length as to not interfere with incorporation into a nucleic acid sequence.
  • the linker can be used for any useful purpose, such as to form polynucleotide multimers (e.g., through linkage of two or more chimeric polynucleotides molecules or IVT polynucleotides) or polynucleotides conjugates, as well as to administer a payload, as described herein.
  • linker examples include, but are not limited to, alkyl, alkenyl, alkynyl, amido, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclyl, each of which can be optionally substituted, as described herein.
  • linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene or propylene glycol monomeric units, e.g., diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran polymers and derivatives thereof.
  • Non-limiting examples of a selectively cleavable bond include an amido bond can be cleaved for example by the use of tris(2-carboxyethyl)phosphine (TCEP), or other reducing agents, and/or photolysis, as well as an ester bond can be cleaved for example by acidic or basic hydrolysis.
  • Methods of Administration can include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering a composition to a subject.
  • a method of administration can be selected to target delivery (e.g., to specifically deliver) to a specific region or system of a body.
  • Modified refers to a changed state or structure of a molecule of the invention. Molecules can be modified in many ways including chemically, structurally, and functionally.
  • the mRNA Attorney Docket No.45817-0124WO1 / MTX959.20 molecules of the present invention are modified by the introduction of non-natural nucleosides and/or nucleotides, e.g., as it relates to the natural ribonucleotides A, U, G, and C.
  • Noncanonical nucleotides such as the cap structures are not considered “modified” although they differ from the chemical structure of the A, C, G, U ribonucleotides.
  • Nanoparticle Composition is a composition comprising one or more lipids. Nanoparticle compositions are typically sized on the order of micrometers or smaller and can include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less.
  • Naturally occurring As used herein, "naturally occurring” means existing in nature without artificial aid.
  • Nucleic acid sequence The terms “nucleic acid sequence,” “nucleotide sequence,” or “polynucleotide sequence” are used interchangeably and refer to a contiguous nucleic acid sequence. The sequence can be either single stranded or double stranded DNA or RNA, e.g., an mRNA.
  • nucleic acid in its broadest sense, includes any compound and/or substance that comprises a polymer of nucleotides. These polymers are often referred to as polynucleotides.
  • nucleic acids or polynucleotides of the invention include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a ⁇ - D-ribo configuration, ⁇ -LNA having an ⁇ -L-ribo configuration (a diastereomer of LNA), 2′- amino-LNA having a 2′-amino functionalization, and 2′-amino- ⁇ -LNA having a 2′- amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or hybrids or combinations thereof.
  • RNAs ribonucleic acids
  • DNAs deoxyribonucleic acids
  • TAAs threose nucleic acids
  • nucleotide sequence encoding refers to the nucleic acid (e.g., an mRNA or DNA molecule) coding sequence which encodes a polypeptide.
  • the coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid Attorney Docket No.45817-0124WO1 / MTX959.20 is administered.
  • the coding sequence can further include sequences that encode signal peptides.
  • Operably linked refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties or the like.
  • Optionally substituted e.g., optionally substituted alkyl
  • X optionally substituted
  • alkyl wherein said alkyl is optionally substituted
  • Part As used herein, a "part" or “region" of a polynucleotide is defined as any portion of the polynucleotide that is less than the entire length of the polynucleotide.
  • Patient refers to a subject who can seek or be in need of treatment, requires treatment, is receiving treatment, will receive treatment, or a subject who is under care by a trained professional for a particular disease or condition. In some embodiments, the treatment is needed, required, or received to prevent or decrease the risk of developing acute disease, i.e., it is a prophylactic treatment.
  • compositions are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable excipients refers any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being substantially nontoxic and non- inflammatory in a patient.
  • Excipients can include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, Attorney Docket No.45817-0124WO1 / MTX959.20 suspension or dispersing agents, sweeteners, and waters of hydration.
  • excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C,
  • compositions described herein also includes pharmaceutically acceptable salts of the compounds described herein.
  • pharmaceutically acceptable salts refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form (e.g., by reacting the free base group with a suitable organic acid).
  • examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzene sulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2- hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, ole
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, Attorney Docket No.45817-0124WO1 / MTX959.20 tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
  • the pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • the pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods.
  • such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used.
  • nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used.
  • Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17 th ed., Mack Publishing Company, Easton, Pa., 1985, p.1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G.
  • solvates means a compound of the invention wherein molecules of a suitable solvent are incorporated in the crystal lattice.
  • a suitable solvent is physiologically tolerable at the dosage administered.
  • solvates can be prepared by crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a mixture thereof.
  • solvents examples include ethanol, water (for example, mono-, di-, and tri-hydrates), N- methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N,N'-dimethylformamide (DMF), N,N'-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like.
  • NMP N- methylpyrrolidinone
  • DMSO dimethyl sulfoxide
  • DMF N,N'-dimethylformamide
  • DMAC N,N'-dimethylacetamide
  • DMEU 1,3-dimethyl-2-imidazolidinone
  • Pharmacokinetic refers to any one or more properties of a molecule or compound as it relates to the determination of the fate of substances administered to a living organism. Pharmacokinetics is divided into several areas including the extent and rate of absorption, distribution, metabolism and excretion.
  • ADME This is commonly referred to as ADME where: (A) Absorption is the Attorney Docket No.45817-0124WO1 / MTX959.20 process of a substance entering the blood circulation; (D) Distribution is the dispersion or dissemination of substances throughout the fluids and tissues of the body; (M) Metabolism (or Biotransformation) is the irreversible transformation of parent compounds into daughter metabolites; and (E) Excretion (or Elimination) refers to the elimination of the substances from the body. In rare cases, some drugs irreversibly accumulate in body tissue.
  • Polynucleotide refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single- stranded deoxyribonucleic acid ("DNA”), as well as triple-, double- and single- stranded ribonucleic acid (“RNA”). It also includes modified, for example by alkylation, and/or by capping, and unmodified forms of the polynucleotide.
  • DNA triple-, double- and single- stranded deoxyribonucleic acid
  • RNA triple-, double- and single- stranded ribonucleic acid
  • polynucleotide includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids "PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA.
  • PNAs peptide nucleic acids
  • the polynucleotide comprises an mRNA.
  • the mRNA is a synthetic mRNA.
  • the synthetic mRNA comprises at least one unnatural nucleobase.
  • all nucleobases of a certain class have been replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with an unnatural nucleobase, e.g., 5-methoxyuridine).
  • the polynucleotide (e.g., a synthetic RNA or a synthetic DNA) comprises only natural nucleobases, i.e., A (adenosine), G (guanosine), C (cytidine), and T (thymidine) in the case of a synthetic DNA, or A, C, G, and U (uridine) in the case of a synthetic RNA.
  • A adenosine
  • G guanosine
  • C cytidine
  • T thymidine
  • A, C, G, and U uridine
  • a codon-nucleotide sequence disclosed herein in DNA form e.g., a vector or an in-vitro translation (IVT) template
  • IVT in-vitro translation
  • both codon-optimized DNA sequences (comprising T) and their corresponding mRNA sequences (comprising U) are considered codon-optimized nucleotide sequence of the present invention.
  • equivalent codon-maps can be generated by replaced one or more bases with non- natural bases.
  • Standard A-T and G-C base pairs form under conditions which allow the formation of hydrogen bonds between the N3-H and C4-oxy of thymidine and the N1 and C6-NH2, respectively, of adenosine and between the C2-oxy, N3 and C4-NH2, of cytidine and the C2-NH2, N′—H and C6-oxy, respectively, of guanosine.
  • guanosine (2-amino-6-oxy-9- ⁇ -D-ribofuranosyl-purine) can be modified to form isoguanosine (2-oxy-6-amino-9- ⁇ -D-ribofuranosyl-purine).
  • Such modification results in a nucleoside base which will no longer effectively form a standard base pair with cytosine.
  • Nonnatural base pairs can be synthesized by the method described in Piccirilli et al., 1990, Nature 343:33-37, for the synthesis of 2,6- diaminopyrimidine and its complement (1-methylpyrazolo-[4,3]pyrimidine-5,7- (4H,6H)-dione.
  • Other such modified nucleotide units which form unique base pairs Attorney Docket No.45817-0124WO1 / MTX959.20 are known, such as those described in Leach et al. (1992) J. Am. Chem. Soc. 114:3675-3683 and Switzer et al., supra.
  • Polypeptide The terms "polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer can comprise modified amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine
  • amino acid including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine
  • the term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function.
  • Polypeptides include encoded polynucleotide products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.
  • a polypeptide can be a monomer or can be a multi-molecular complex such as a dimer, trimer or tetramer. They can also comprise single chain or multichain polypeptides. Most commonly disulfide linkages are found in multichain polypeptides.
  • the term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.
  • a "peptide" can be less than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
  • Polypeptide variant refers to molecules that differ in their amino acid sequence from a native or reference sequence.
  • the amino acid sequence variants can possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence.
  • variants will possess at least about 50% identity, at least about 60% identity, at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 99% Attorney Docket No.45817-0124WO1 / MTX959.20 identity to a native or reference sequence.
  • the term "preventing" refers to partially or completely delaying onset of an infection, disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying progression from an infection, a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and/or condition.
  • Prophylactic As used herein, “prophylactic” refers to a therapeutic or course of action used to prevent the spread of disease.
  • Prophylaxis As used herein, a “prophylaxis” refers to a measure taken to maintain health and prevent the spread of disease.
  • An “immune prophylaxis” refers to a measure to produce active or passive immunity to prevent the spread of disease.
  • Pseudouridine As used herein, pseudouridine ( ⁇ ) refers to the C- glycoside isomer of the nucleoside uridine.
  • a "pseudouridine analog” is any modification, variant, isoform or derivative of pseudouridine.
  • pseudouridine analogs include but are not limited to 1-carboxymethyl-pseudouridine, 1-propynyl-pseudouridine, 1-taurinomethyl-pseudouridine, 1-taurinomethyl-4-thio- pseudouridine, 1-methylpseudouridine (m 1 ⁇ ) (also known as N1-methyl- pseudouridine), 1-methyl-4-thio-pseudouridine (m 1 s 4 ⁇ ), 4-thio-1-methyl- pseudouridine, 3-methyl-pseudouridine (m 3 ⁇ ), 2-thio-1-methyl-pseudouridine, 1- methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4- thio-uridine, 4-methoxy-pseu
  • Reference Nucleic Acid Sequence refers to a starting nucleic acid sequence (e.g., a RNA, e.g., an mRNA sequence) that can be sequence optimized. In some embodiments, the reference nucleic acid sequence is a wild type nucleic acid sequence, a fragment or a variant thereof.
  • the reference nucleic acid sequence is a previously sequence optimized nucleic acid sequence.
  • Salts In some aspects, the pharmaceutical composition for delivery disclosed herein and comprises salts of some of their lipid constituents. The term “salt” includes any anionic and cationic complex.
  • Non-limiting examples of anions include inorganic and organic anions, e.g., fluoride, chloride, bromide, iodide, oxalate (e.g., hemioxalate), phosphate, phosphonate, hydrogen phosphate, dihydrogen phosphate, oxide, carbonate, bicarbonate, nitrate, nitrite, nitride, bisulfite, sulfide, sulfite, bisulfate, sulfate, thiosulfate, hydrogen sulfate, borate, formate, acetate, benzoate, citrate, tartrate, lactate, acrylate, polyacrylate, fumarate, maleate, itaconate, glycolate, gluconate, malate, mandelate, tiglate, ascorbate, salicylate, polymethacrylate, perchlorate, chlorate, chlorite, hypochlorite, bromate, hypobromite, iodate
  • sample refers to a subset of its tissues, cells or component parts (e.g., body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).
  • body fluids including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).
  • a sample further can include a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs.
  • a sample further refers to a medium, such as a nutrient broth or gel, which can contain cellular components, such as proteins or nucleic acid molecule.
  • Signal sequence As used herein, the phrases "signal sequence,” “signal peptide,” and “transit peptide” are used interchangeably and refer to a sequence that can direct the transport or localization of a protein to a certain organelle, cell compartment, or extracellular export. The term encompasses both the signal sequence polypeptide and the nucleic acid sequence encoding the signal sequence. Thus, references to a signal sequence in the context of a nucleic acid refer in fact to the nucleic acid sequence encoding the signal sequence polypeptide.
  • Similarity refers to the overall relatedness between polymeric molecules, e.g. between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as is understood in the art.
  • Single unit dose As used herein, a “single unit dose” is a dose of any therapeutic administered in one dose/at one time/single route/single point of contact, i.e., single administration event.
  • Specific delivery means delivery of more (e.g., at least 1.5 fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold more) of a polynucleotide by a nanoparticle to a target tissue of interest (e.g., mammalian liver) compared to an off-target tissue (e.g., mammalian spleen).
  • a target tissue of interest e.g., mammalian liver
  • off-target tissue e.g., mammalian spleen
  • the level of delivery of a nanoparticle to a particular tissue can be measured by comparing the amount of protein produced in a tissue to the weight of said tissue, comparing the amount of polynucleotide in a tissue to the weight of said tissue, comparing the amount of protein produced in a tissue to the amount of total protein in said tissue, or comparing the amount of polynucleotide in a tissue to the amount of total polynucleotide in said tissue.
  • a polynucleotide is specifically provided to a mammalian kidney as compared to the liver and spleen if 1.5, 2-fold, 3-fold, 5-fold, 10-fold, 15 fold, or 20 fold more polynucleotide per 1 g of tissue is delivered to a kidney compared to that delivered to Attorney Docket No.45817-0124WO1 / MTX959.20 the liver or spleen following systemic administration of the polynucleotide.
  • a surrogate such as an animal model (e.g., a rat model).
  • Stable As used herein “stable” refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and in some cases capable of formulation into an efficacious therapeutic agent. [00859] Stabilized: As used herein, the term “stabilize,” “stabilized,” “stabilized region” means to make or become stable. [00860] Subject: By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
  • Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; bears, food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on.
  • pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs
  • the mammal is a human subject.
  • a subject is a human patient.
  • a subject is a human patient in need of treatment.
  • Substantially refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • One of ordinary skill in the biological arts will understand that biological and chemical characteristics rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical characteristics.
  • an individual who is susceptible to a disease, disorder, and/or condition can be characterized by one or more of the following: (1) a genetic mutation associated with development of the disease, disorder, and/or condition; (2) a genetic polymorphism associated with development of the disease, disorder, and/or condition; (3) increased and/or decreased expression and/or activity of a protein and/or nucleic acid associated with the disease, disorder, and/or condition; (4) habits and/or lifestyles associated with development of the disease, disorder, and/or condition; (5) a family history of the disease, disorder, and/or condition; and (6) exposure to and/or infection with a microbe associated with development of the disease, disorder, and/or condition.
  • an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
  • Sustained release As used herein, the term “sustained release” refers to a pharmaceutical composition or compound release profile that conforms to a release rate over a specific period of time.
  • Synthetic The term “synthetic” means produced, prepared, and/or manufactured by the hand of man. Synthesis of polynucleotides or other molecules of the present invention can be chemical or enzymatic.
  • Targeted cells refers to any one or more cells of interest.
  • the cells can be found in vitro, in vivo, in situ or in the tissue or organ of an organism.
  • the organism can be an animal, for example a mammal, a human, a subject or a patient.
  • Target tissue refers to any one or more tissue types of interest in which the delivery of a polynucleotide would result in a desired biological and/or pharmacological effect. Examples of target tissues of Attorney Docket No.45817-0124WO1 / MTX959.20 interest include specific tissues, organs, and systems or groups thereof.
  • a target tissue can be a liver, a kidney, a lung, a spleen, or a vascular endothelium in vessels (e.g., intra-coronary or intra-femoral).
  • An “off-target tissue” refers to any one or more tissue types in which the expression of the encoded protein does not result in a desired biological and/or pharmacological effect.
  • the presence of a therapeutic agent in an off-target issue can be the result of: (i) leakage of a polynucleotide from the administration site to peripheral tissue or distant off-target tissue via diffusion or through the bloodstream (e.g., a polynucleotide intended to express a polypeptide in a certain tissue would reach the off-target tissue and the polypeptide would be expressed in the off-target tissue); or (ii) leakage of an polypeptide after administration of a polynucleotide encoding such polypeptide to peripheral tissue or distant off-target tissue via diffusion or through the bloodstream (e.g., a polynucleotide would expressed a polypeptide in the target tissue, and the polypeptide would diffuse to peripheral tissue).
  • Targeting sequence refers to a sequence that can direct the transport or localization of a protein or polypeptide.
  • Therapeutic Agent refers to an agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.
  • an mRNA encoding a Snu13 polypeptide can be a therapeutic agent.
  • therapeutically effective amount means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.
  • an agent to be delivered e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.
  • Therapeutically effective outcome means an outcome that is sufficient in a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.
  • Transcription refers to methods to produce mRNA (e.g., an mRNA sequence or template) from DNA (e.g., a DNA template or sequence).
  • Transfection refers to the introduction of a polynucleotide (e.g., exogenous nucleic acids) into a cell wherein a polypeptide encoded by the polynucleotide is expressed (e.g., mRNA) or the polypeptide modulates a cellular function (e.g., siRNA, miRNA).
  • expression of a nucleic acid sequence refers to translation of a polynucleotide (e.g., an mRNA) into a polypeptide or protein and/or post-translational modification of a polypeptide or protein.
  • Treating, treatment, therapy refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a disease.
  • treating a disease can refer to diminishing symptoms associate with the disease, prolong the lifespan (increase the survival rate) of patients, reducing the severity of the disease, preventing or delaying the onset of the disease, etc.
  • Unmodified refers to any substance, compound or molecule prior to being changed in some way. Unmodified can, but does not always, refer to the wild type or native form of a biomolecule. Molecules can undergo a series of modifications whereby each modified molecule can serve as the "unmodified" starting molecule for a subsequent modification.
  • Uracil is one of the four nucleobases in the nucleic acid of RNA, and it is represented by the letter U.
  • Uracil can be attached to a ribose ring, or more specifically, a ribofuranose via a ⁇ -N 1 -glycosidic bond to yield the nucleoside uridine.
  • the nucleoside uridine is also commonly abbreviated according to the one letter code of its nucleobase, i.e., U.
  • Uridine content when a Attorney Docket No.45817-0124WO1 / MTX959.20 monomer in a polynucleotide sequence is U, such U is designated interchangeably as a "uracil” or a “uridine.”
  • Uridine content The terms "uridine content” or "uracil content” are interchangeable and refer to the amount of uracil or uridine present in a certain nucleic acid sequence. Uridine content or uracil content can be expressed as an absolute value (total number of uridine or uracil in the sequence) or relative (uridine or uracil percentage respect to the total number of nucleobases in the nucleic acid sequence).
  • Uridine-Modified Sequence refers to a sequence optimized nucleic acid (e.g., a synthetic mRNA sequence) with a different overall or local uridine content (higher or lower uridine content) or with different uridine patterns (e.g., gradient distribution or clustering) with respect to the uridine content and/or uridine patterns of a candidate nucleic acid sequence.
  • uridine-modified sequence and uracil- modified sequence
  • a "high uridine codon” is defined as a codon comprising two or three uridines
  • a "low uridine codon” is defined as a codon comprising one uridine
  • a "no uridine codon” is a codon without any uridines.
  • a uridine- modified sequence comprises substitutions of high uridine codons with low uridine codons, substitutions of high uridine codons with no uridine codons, substitutions of low uridine codons with high uridine codons, substitutions of low uridine codons with no uridine codons, substitution of no uridine codons with low uridine codons, substitutions of no uridine codons with high uridine codons, and combinations thereof.
  • a high uridine codon can be replaced with another high uridine codon.
  • a low uridine codon can be replaced with another low uridine codon.
  • a no uridine codon can be replaced with another no uridine codon.
  • a uridine-modified sequence can be uridine enriched or uridine rarefied.
  • Uridine Enriched As used herein, the terms "uridine enriched" and grammatical variants refer to the increase in uridine content (expressed in absolute value or as a percentage value) in a sequence optimized nucleic acid (e.g., a synthetic mRNA sequence) with respect to the uridine content of the corresponding candidate Attorney Docket No.45817-0124WO1 / MTX959.20 nucleic acid sequence.
  • Uridine enrichment can be implemented by substituting codons in the candidate nucleic acid sequence with synonymous codons containing less uridine nucleobases. Uridine enrichment can be global (i.e., relative to the entire length of a candidate nucleic acid sequence) or local (i.e., relative to a subsequence or region of a candidate nucleic acid sequence).
  • Uridine Rarefied As used herein, the terms "uridine rarefied" and grammatical variants refer to a decrease in uridine content (expressed in absolute value or as a percentage value) in a sequence optimized nucleic acid (e.g., a synthetic mRNA sequence) with respect to the uridine content of the corresponding candidate nucleic acid sequence.
  • Uridine rarefication can be implemented by substituting codons in the candidate nucleic acid sequence with synonymous codons containing less uridine nucleobases. Uridine rarefication can be global (i.e., relative to the entire length of a candidate nucleic acid sequence) or local (i.e., relative to a subsequence or region of a candidate nucleic acid sequence).
  • Variant The term variant as used in present disclosure refers to both natural variants (e.g., polymorphisms, isoforms, etc.) and artificial variants in which at least one amino acid residue in a native or starting sequence (e.g., a wild type sequence) has been removed and a different amino acid inserted in its place at the same position.
  • substitutional variants can be single, where only one amino acid in the molecule has been substituted, or they can be multiple, where two or more amino acids have been substituted in the same molecule. If amino acids are inserted or deleted, the resulting variant would be an "insertional variant” or a “deletional variant” respectively.
  • initiation codon used interchangeably with the term “start codon”, refers to the first codon of an open reading frame that is translated by the ribosome and is comprised of a triplet of linked adenine-uracil-guanine nucleobases.
  • the initiation codon is depicted by the first letter codes of adenine (A), uracil (U), and guanine (G) and is often written simply as “AUG”. Although natural mRNAs may use codons other than AUG as the initiation codon, which are referred to herein as “alternative initiation codons”, the initiation codons of polynucleotides described herein use the AUG codon.
  • the sequence comprising the initiation codon is recognized via Attorney Docket No.45817-0124WO1 / MTX959.20 complementary base-pairing to the anticodon of an initiator tRNA (Met-tRNAi Met ) bound by the ribosome.
  • Open reading frames may contain more than one AUG initiation codon, which are referred to herein as “alternate initiation codons”.
  • the initiation codon plays a critical role in translation initiation.
  • the initiation codon is the first codon of an open reading frame that is translated by the ribosome.
  • the initiation codon comprises the nucleotide triplet AUG, however, in some instances translation initiation can occur at other codons comprised of distinct nucleotides.
  • RNA-RNA interactions between messenger RNA molecules (mRNAs), the 40S ribosomal subunit, other components of the translation machinery (e.g., eukaryotic initiation factors; eIFs).
  • mRNAs messenger RNA molecules
  • eIFs eukaryotic initiation factors
  • the current model of mRNA translation initiation postulates that the pre-initiation complex (alternatively “43S pre-initiation complex”; abbreviated as “PIC”) translocates from the site of recruitment on the mRNA (typically the 5′ cap) to the initiation codon by scanning nucleotides in a 5′ to 3′ direction until the first AUG codon that resides within a specific translation-promotive nucleotide context (the Kozak sequence) is encountered (Kozak (1989) J Cell Biol 108:229-241).
  • PIC pre-initiation complex
  • Kozak sequence refers to a translation initiation enhancer element to enhance expression of a gene or open reading frame, and which in eukaryotes, is located in the 5′ UTR.
  • Polynucleotides disclosed herein comprise a Kozak consensus sequence, or a derivative or modification thereof.
  • Modified refers to a changed state or a change in composition or structure of a polynucleotide (e.g., mRNA).
  • Polynucleotides may be modified in various ways including chemically, structurally, and/or functionally.
  • polynucleotides may be structurally modified by the incorporation of one or more RNA elements, wherein the RNA element comprises a sequence and/or an RNA secondary structure(s) that provides one or more functions (e.g., translational regulatory activity).
  • RNA element comprises a sequence and/or an RNA secondary structure(s) that provides one or more functions (e.g., translational regulatory activity).
  • polynucleotides of the disclosure may be comprised of one or more modifications (e.g., may include one or more chemical, structural, or functional modifications, including any combination thereof).
  • nucleobase refers to a purine or pyrimidine heterocyclic compound found in nucleic acids, including any derivatives or analogs of the naturally occurring purines and pyrimidines that confer improved properties (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof.
  • Adenine, cytosine, guanine, thymine, and uracil are the nucleobases predominately found in natural nucleic acids.
  • nucleobase sequence of a SEQ ID NO described herein encompasses both natural nucleobases and chemically modified nucleobases (e.g., a “U” designation in a SEQ ID NO encompasses both uracil and chemically modified uracil).
  • nucleoside refers to a compound containing a sugar molecule (e.g., a ribose in RNA or a deoxyribose in DNA), or derivative or analog thereof, covalently linked to a nucleobase (e.g., a purine or pyrimidine), or a derivative or analog thereof (also referred to herein as “nucleobase”), but lacking an internucleoside linking group (e.g., a phosphate group).
  • a sugar molecule e.g., a ribose in RNA or a deoxyribose in DNA
  • nucleobase e.g., a purine or pyrimidine
  • nucleobase also referred to herein as “nucleobase”
  • an internucleoside linking group e.g., a phosphate group
  • nucleotide refers to a nucleoside covalently bonded to an internucleoside linking group (e.g., a phosphate group), or any derivative, analog, or modification thereof that confers improved chemical and/or functional properties Attorney Docket No.45817-0124WO1 / MTX959.20 (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof.
  • Nucleic acid As used herein, the term “nucleic acid” is used in its broadest sense and encompasses any compound and/or substance that includes a polymer of nucleotides, or derivatives or analogs thereof.
  • nucleic acid and “polynucleotide” are equivalent and are used interchangeably.
  • exemplary nucleic acids or polynucleotides of the disclosure include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), DNA-RNA hybrids, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, mRNAs, modified mRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a ⁇ -D-ribo configuration, ⁇ -LNA having an ⁇ -L-ribo configuration (a di
  • nucleic acid structure refers to the arrangement or organization of atoms, chemical constituents, elements, motifs, and/or sequence of linked nucleotides, or derivatives or analogs thereof, that comprise a nucleic acid (e.g., an mRNA). The term also refers to the two-dimensional or three-dimensional state of a nucleic acid.
  • RNA structure refers to the arrangement or organization of atoms, chemical constituents, elements, motifs, and/or sequence of linked nucleotides, or derivatives or analogs thereof, comprising an RNA molecule (e.g., an mRNA) and/or refers to a two-dimensional and/or three dimensional state of an RNA molecule.
  • Nucleic acid structure can be further demarcated into four organizational categories referred to herein as “molecular structure”, “primary structure”, “secondary structure”, and “tertiary structure” based on increasing organizational complexity.
  • Open Reading Frame As used herein, the term “open reading frame”, abbreviated as “ORF”, refers to a segment or region of an mRNA molecule that encodes a polypeptide.
  • the ORF comprises a continuous stretch of non-overlapping, Attorney Docket No.45817-0124WO1 / MTX959.20 in-frame codons, beginning with the initiation codon and ending with a stop codon, and is translated by the ribosome.
  • Pre-Initiation Complex As used herein, the term “pre-initiation complex” (alternatively “43S pre-initiation complex”; abbreviated as “PIC”) refers to a ribonucleoprotein complex comprising a 40S ribosomal subunit, eukaryotic initiation factors (eIF1, eIF1A, eIF3, eIF5), and the eIF2-GTP-Met-tRNA i Met ternary complex, that is intrinsically capable of attachment to the 5′ cap of an mRNA molecule and, after attachment, of performing ribosome scanning of the 5′ UTR.
  • pre-initiation complex refers to a ribonucleoprotein complex comprising a 40S ribosomal subunit, eukaryotic initiation factors (eIF1, eIF1A, eIF3, eIF5), and the eIF2-GTP-Met-tRNA i Met ternary complex, that is intrinsically capable of attachment to the 5′ cap of an mRNA
  • RNA element refers to a portion, fragment, or segment of an RNA molecule that provides a biological function and/or has biological activity (e.g., translational regulatory activity). Modification of a polynucleotide by the incorporation of one or more RNA elements, such as those described herein, provides one or more desirable functional properties to the modified polynucleotide.
  • RNA elements, as described herein can be naturally-occurring, non- naturally occurring, synthetic, engineered, or any combination thereof.
  • naturally-occurring RNA elements that provide a regulatory activity include elements found throughout the transcriptomes of viruses, prokaryotic and eukaryotic organisms (e.g., humans).
  • RNA elements in particular eukaryotic mRNAs and translated viral RNAs have been shown to be involved in mediating many functions in cells.
  • exemplary natural RNA elements include, but are not limited to, translation initiation elements (e.g., internal ribosome entry site (IRES), see Kieft et al., (2001) RNA 7(2):194-206), translation enhancer elements (e.g., the APP mRNA translation enhancer element, see Rogers et al., (1999) J Biol Chem 274(10):6421-6431), mRNA stability elements (e.g., AU-rich elements (AREs), see Garneau et al., (2007) Nat Rev Mol Cell Biol 8(2):113-126), translational repression element (see e.g., Blumer et al., (2002) Mech Dev 110(1-2):97-112), protein-binding RNA elements (e.g., iron- responsive element, see Selezneva et al.
  • Residence time refers to the time of occupancy of a pre-initiation complex (PIC) or a ribosome at a discrete position or location along an mRNA molecule.
  • translational regulatory activity refers to a biological function, mechanism, or process that modulates (e.g., regulates, influences, controls, varies) the activity of the translational apparatus, including the activity of the PIC and/or ribosome.
  • the desired translation regulatory activity promotes and/or enhances the translational fidelity of mRNA translation. In some aspects, the desired translational regulatory activity reduces and/or inhibits leaky scanning. 27. Equivalents and Scope [00898] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims. In the claims, articles such as "a,” “an,” and “the” can mean one or more than one unless indicated to the contrary or otherwise evident from the context.
  • Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. [00899] It is also noted that the term “comprising" is intended to be open and permits but does not require the inclusion of additional elements or steps.
  • compositions of the invention e.g., any nucleic acid or protein encoded thereby; any method of production; any method of use; etc.
  • Any particular embodiment of the compositions of the invention can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.
  • All cited sources for example, references, publications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control.
  • Exemplary sequence optimized nucleotide sequence encoding Snu13 variants are provided in SEQ ID NOs:300-317. Attorney Docket No.45817-0124WO1 / MTX959.20 [00907]
  • the mRNA sequence includes both 5′ and 3′ UTR regions flanking the ORF sequence (nucleotide).
  • the 5′ UTR and 3′ UTR sequences are SEQ ID NOs:50 and 108, respectively.
  • modified mRNA can be generated using N1-methylpseudouridine-5′-Triphosphate to ensure that the mRNAs contain 100% N1-methylpseudouridine instead of uridine.
  • modified mRNA can be generated using N1-methoxyuridine-5′- Triphosphate to ensure that the mRNAs contain 100% 5-methoxyuridine instead of uridine.
  • Snu13 variant mRNA can be synthesized with a primer that introduces a polyA-tail, and a cap structure is generated on both mRNAs using co- transcriptional capping via m 7 G-ppp-Gm-AG tetranucleotide to incorporate a m 7 G- ppp-Gm-AG 5′ cap1.
  • Snu13 variant mRNA can be synthesized and the polyA-tail introduced during Gibson assembly of the DNA template.
  • EXAMPLE 2 Production of Nanoparticle Compositions A.
  • Nanoparticles can be made with mixing processes such as microfluidics and T-junction mixing of two fluid streams, one of which contains the polynucleotide and the other has the lipid components.
  • Lipid compositions are prepared by combining an ionizable amino lipid disclosed herein, e.g., a lipid according to Formula (I) such as Compound II or a lipid according to Formula (III) such as Compound VI, a phospholipid (such as DOPE or Attorney Docket No.45817-0124WO1 / MTX959.20 DSPC, obtainable from Avanti Polar Lipids, Alabaster, AL), a PEG lipid (such as 1,2 dimyristoyl sn glycerol methoxypolyethylene glycol, also known as PEG-DMG, obtainable from Avanti Polar Lipids, Alabaster, AL), and a structural lipid (such as cholesterol, obtainable from Sigma Aldrich,
  • Nanoparticle compositions including a polynucleotide and a lipid composition are prepared by combining the lipid solution with a solution including the a polynucleotide at lipid composition to polynucleotide wt:wt ratios between about 5:1 and about 50:1.
  • NanoAssemblr microfluidic based system at flow rates between about 10 ml/min and about 18 ml/min into the polynucleotide solution to produce a suspension with a water to ethanol ratio between about 1:1 and about 4:1.
  • solutions of the RNA at concentrations of 0.1 mg/ml in deionized water are diluted in 50 mM sodium citrate buffer at a pH between 3 and 4 to form a stock solution.
  • Nanoparticle compositions can be processed by dialysis to remove ethanol and achieve buffer exchange.

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Abstract

La présente divulgation concerne des polypeptides Snu13 et des polynucléotides (p. ex., un ARNm) codant pour ceux-ci destinés à être utilisés dans la commande de traduction d'un second polynucléotide (p. ex., un ARNm cible). Des thérapies par ARNm de la divulgation expriment des polypeptides Snu13, qui commandent la traduction d'un polynucléotide cible comprenant un site de fixation Snu13. Ainsi, la divulgation concerne également des compositions ou des systèmes et des utilisations associées, consistant en un premier polynucléotide codant pour une molécule cible et consistant en un site de fixation Snu13 et un second polynucléotide codant pour un polypeptide Snu13. La divulgation concerne également des agents d'administration (p. ex., des nanoparticules lipidiques) et des compositions comprenant les polynucléotides Snu13 (p. ex., des ARNm) de la divulgation.
PCT/US2023/085370 2022-12-22 2023-12-21 Polypeptides de petite ribonucléoprotéine nucléaire 13, polynucléotides et utilisations associées WO2024137953A2 (fr)

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